Compositions and methods for treating glioblastoma by modulating a mgmt enhancer

ABSTRACT

The present invention provides, inter alia, methods for treating or ameliorating the effects of a cancer in a subject, e.g., glioblastoma (GBM) in a subject that is resistant to temozolomide (TMZ), methods for delaying the emergence of TMZ resistance and/or increasing the TMZ sensitivity in a subject, and methods for reducing tumor aggressiveness in a subject. Also provided are pharmaceutical compositions and kits to implement such methods.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of PCT international application no. PCT/US2019/028366, filed on Apr. 19, 2019, which claims benefit of U.S. Provisional Patent Application Ser. No. 62/660,395, filed on Apr. 20, 2018 which applications are incorporated by reference herein in their entireties.

GOVERNMENT FUNDING

This invention was made with government support under CA157489 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file “CU18203-seq.txt”, file size of 9 KB, created on Oct. 2, 2020. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).

FIELD OF INVENTION

The present invention provides, inter alia, methods for treating or ameliorating the effects of a cancer in a subject, e.g., glioblastoma (GBM) in a subject that is resistant to temozolomide (TMZ), methods for delaying the emergence of TMZ resistance and/or increasing the TMZ sensitivity in a subject, and methods for reducing tumor aggressiveness in a subject. Also provided are pharmaceutical compositions and kits to implement such methods.

BACKGROUND OF THE INVENTION

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Despite current therapy, median survival for GBM patients is approximately 15 months (Stupp et al. 2005). Temozolomide (TMZ) has been the standard chemotherapy for newly diagnosed GBM for more than a decade (Stupp et al. 2005). However, almost all patients eventually develop resistance. Moreover, recurrent tumors in general are more aggressive than primary tumors. Therefore, there is a critical need to understand how TMZ resistance is acquired and whether there is any connection between TMZ resistance and tumor aggressiveness.

While multiple factors have been associated with TMZ resistance, expression of O₆-methylguanine-DNA-methytransferase (MGMT) remains a major cause (Kitange et al. 2009). MGMT is a DNA repair protein, which removes the cytotoxic O6-methylguanine (O₆MG) DNA lesions generated by TMZ, and high MGMT expression in cells is mechanistically linked to robust TMZ resistance. MGMT expression is regulated by the methylation of a promoter/enhancer (P/E) region, which contains a promoter and a 59 bp cis-acting enhancer element that spans the first exon-intron boundary of MGMT gene (Everhard et al. 2009). Several large cohort studies indicate an association between DNA methylation of the (P/E) region and favorable outcomes of TMZ treatment (Moradi et al. 2017; Brandes et al. 2017). This observation led to the hypothesis that MGMT inhibition may be a plausible strategy for sensitizing TMZ therapy in MGMT expressed tumors (Lavon et al. 2007; Wickstrom et al. 2015; Bobustuc et al. 2010; Taspinar et al. 2013). However, combinations of TMZ with MGMT inhibitors such as O₆-benzylguanine (O₆BG), a synthetic derivative of guanine that inactivates MGMT, resulted in enhanced hematologic toxicities, a reduced therapeutic window and no clinical benefit compared to TMZ alone (Quinn et al. 2009; Blumenthal et al. 2015; Warren et al. 2012). Moreover, there is significant discordance between promoter methylation status and MGMT protein expression in GBM with wild-type MGMT coding sequence (Park et al. 2012; Kreth et al. 2011; Hegi et al. 2005; Xie et al. 2011; Lund et al. 2017). These observations indicate that in addition to promoter methylation, other factors may regulate MGMT expression and confer TMZ resistance, and that identification of these additional mechanisms of MGMT regulation may provide a strong rationale for the development of a new class of drugs for this deadly disease.

Accordingly, there is a need for a new therapeutic strategy that can improve the result of TMZ treatment by delaying the emergence of TMZ resistance and/or increasing the TMZ sensitivity in a subject in need thereof. This invention is directed to meet these and other needs.

SUMMARY OF THE INVENTION

Besides DNA methylation, post translational modifications on histone proteins also regulate gene expression. Distinct histone modifications are found at gene regulatory elements important for gene transcription (Bannister et al. 2011). For example, H3K4me3 is enriched at promoters of active genes, whereas H3K27me3 is enriched at promoters of repressed genes. In addition to promoters, enhancers, a DNA element that promotes the transcription of regulated genes via a long-range interaction with their cognate promoters, are also surrounded by nucleosomes with distinct modifications on histones (Banerji et al. 1981). Enhancers in general can be classified as active, primed or poised ones based on histone modifications of surrounding nucleosomes. Active enhancers are typically surrounded by nucleosomes with H3K4me1 and H3K27ac (Shlyueva et al. 2014). Primed enhancers are marked with H3K4me1 and a lack of histone acetylation, and poised enhancers are additionally marked by H3K27me3, a repressive mark (Heinz et al. 2015).

Without being bound to a particular theory, this invention relates to the identification of a novel distal enhancer, named K-M enhancer that is situated between MKI67 and MGMT promoters and is 560 kb away from the MGMT promoter. The K-M enhancer activates MGMT gene expression even in the presence of a hypermethylated promoter. Moreover, the K-M enhancer also regulates the expression of Ki67. Ki67 is a well-known proliferation mark for many tumor types including GBM. Increased Ki67 staining is associated with elevated aggressiveness of glioblastoma (Schlotter et al. 2017; Zhou et al. 2017; Kloppel et al. 2017). This invention shows that deletion of the enhancer results in reduced expression of MGMT as well as Ki67. Moreover, brain tumor cells lacking the enhancer are sensitive to TMZ and exhibit reduced growth rate. Together, these studies uncover a previously unknown mechanism regulating both TMZ resistance and proliferation of GBM cells.

Accordingly, one aspect of the present invention is a method for treating or ameliorating the effects of a cancer in a subject. This method comprises administering to the subject a therapeutically effective amount of a first agent that modulates the activity of an enhancer and a therapeutically effective amount of a second agent that is used to treat the cancer.

Another aspect of the present invention is a method for delaying the emergence of temozolomide (TMZ) resistance and/or increasing the TMZ sensitivity in a subject. This method comprises administering to the subject a therapeutically effective amount of an agent that modulates the activity of an enhancer.

Another aspect of the present invention is a method for reducing tumor aggressiveness in a subject. This method comprises administering to the subject a therapeutically effective amount of an agent that modulates the activity of an enhancer.

An additional aspect of the present invention is a pharmaceutical composition. This pharmaceutical composition comprises 1) a therapeutically effective amount of a first agent that modulates the activity of an enhancer in a subject suffering from a cancer and 2) a therapeutically effective amount of a second agent that is used to treat the cancer.

A further aspect of the present invention is a kit. This kit comprises the aforementioned pharmaceutical composition, and instructions of use.

Yet another aspect of the present invention is a method for treating or ameliorating the effects of gliobastoma (GBM) in a subject that is resistant to temozolomide (TMZ). This method comprises administering to the subject a therapeutically effective amount of a p300 HAT inhibitor or a Bromodomain inhibitor (BETi) and a therapeutically effective amount of TMZ.

Still another aspect of the present invention is a method of modulating proliferation of brain tumor cells with or without activation of a MGMT enhancer. This method comprises contacting the cells with an effective amount of a composition selected from a HAT inhibitor, a p300 inhibitor, a bromodomain inhibitor, and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color. Copies of this patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1I show that histone modifications marking enhancers are altered in a TMZ resistant PDX line.

FIG. 1A is the schematic diagram for the development of 5199 and 3080 xenograft lines from parental GBM12 cells. Mice with GBM12 flank tumors were treated with placebo or three cycles of TMZ (50 mg/kg/d for 5 days every 28 days). Tumors were dissected after reaching >1500 mm³. The xenograft subline established from placebo treated tumor was named 5199 and that from TMZ treated tumor was named 3080.

FIG. 1B shows the analysis of MGMT levels in protein lysates of 5199 and 3080 xenograft tissues by Western blotting. α-tubulin was used as a loading control.

FIG. 1C shows the analysis of MGMT promoter methylation in 5199 versus 3080 xenograft lines by MS-PCR. Universal methylated DNA was used as positive control and normal human brain DNA was used as negative control.

FIG. 1D is the aggregate plot showing the average ChIP-seq reads distribution for the histone mark, H3K4me1, surrounding the published enhancer regions.

FIG. 1E is the aggregate plot showing the average ChIP-seq reads distribution for the histone mark, H3K27ac, surrounding the published enhancer regions.

FIG. 1F is the aggregate plot showing the average ChIP-seq reads for the histone mark, H3K4me3, surrounding transcription start site (TSS).

FIG. 1G is the aggregate plot showing the average ChIP-seq reads for the histone mark, H3K36me3, across gene bodies. TTS: transcription termination site.

FIG. 1H provides heat maps showing cluster analysis, based on H3K27ac and H3K4me1 alterations, and expression change for genes closest to each genomic locus. The ratio of ChIP-seq reads density (3080 reads/5199 reads) for H3K4me1 and H3K27ac was calculated and subsequently analyzed by k-means cluster analysis. Changes in gene expression were calculated as RNA-seq reads ratio (3080 reads/5199 reads). Color scale represents decreased (green) and increased (red) signal intensity (intensity=log₂ 3080 reads/5199 reads).

FIG. 1I provides bar graphs showing Ingenuity Pathway Analysis for Group-1 genes. Pathways with a p-value <0.05 are presented.

FIGS. 2A-2D show the delineation of a putative enhancer associated with MGMT gene in 3080 xenograft subline.

FIG. 2A provides IGV snapshots showing H3K4me1, H3K27ac, H3K4me3, and H3K36me3 ChIP-seq reads density at the putative enhancer region, MGMT promoter and gene body in 5199 (TMZ-sensitive) and 3080 (TMZ resistant) xenograft lines.

FIG. 2B shows H3K4me1 (bottom left) and H3K27ac (bottom right) occupancy at the putative K-M enhancer in 5199 (parental) and 3080 (TMZ resistant) xenograft tumors were analyzed by ChIP-qPCR. Three different sets of primers (PE1-3) were designed to analyze against putative enhancer region. A set of primers amplifying 5 kb away from the putative enhancer (PE+5 kb) was used as negative control. Top: magnified view of H3K27ac ChIP-read peaks and the location of each primer set.

FIG. 2C shows the effect of each fragment on transcription of luciferase reporter gene in 5199 and 3080 cells. Fragments R1-R10 covering putative enhancer region (top) were cloned upstream of luciferase promoter (middle). Each construct were transfected into 5199 or 3080 cells along with a plasmid expressing Renilla luciferase. Firefly and Renilla luciferase activities were measured 36 h after transfection. Luciferase activity was normalized to the Renilla luciferase activity (internal control) first and subsequently normalized to an empty vector control.

FIG. 2D shows enhancer-promoter interaction analyzed by Chromatin Conformation Capture (3C) assay. Relative crosslinking frequency of each restriction fragment (F1-F6) was calculated as described in the experimental procedures and was plotted on genomic location of the 3′ cutting site of each fragment (X-axis). Values reported were derived from three biological repeats (*p<0.05, **p<0.01).

FIGS. 3A-3F show the analysis of the enhancer activity and MGMT expression from three pairs of primary and recurrent GBM patient samples.

FIG. 3A shows the relative occupancy of H3K4me1 in the K-M enhancer region in primary and matched recurrent tumors from three GBM patients was analyzed by ChIP-qPCR using primers described in FIGS. 2A-2D.

FIG. 3B shows the relative occupancy of H3K27ac in the K-M enhancer region in primary and matched recurrent tumors from three GBM patients was analyzed by ChIP-qPCR using primers described in FIGS. 2A-2D.

FIG. 3C shows the MGMT expression level in each tumor was analyzed by immunofluorescence. The MGMT signal (area×average intensity) from 100 individual nuclei was measured and one-way ANOVA was used for the calculation of significance. (****p<0.0001, ns: not significant, n=100)

FIG. 3D provides the analysis of MGMT expression in proliferating tumor cells. Multicolor immunofluorescence was performed using antibodies against CD45, Ki67, and MGMT. Actively dividing tumor cells (Ki67 staining without CD45 staining) were identified and the percentage of MGMT expressing cells within Ki67 positive and CD45 negative population were calculated. Two-tail paired student t-test was used for calculation of significance. (*p<0.05)

FIG. 3E provides representative images showing co-localization of MGMT and Ki67 in tissue sections from primary tumor obtained from patient #1.

FIG. 3F Representative images showing co-localization of MGMT and Ki67 in tissue sections from recurrent tumor obtained from patient #1.

FIGS. 4A-4G show that targeting p300 catalytic domain to the K-M enhancer increases H3K27ac at the enhancer region and elevates MGMT gene transcription.

FIG. 4A is a schematic diagram showing procedure of targeting p300 catalytic domain (p300 core) by CRISPR/dCas9 system. Experimental design involved co-transfection of dCas9^(p300 Core) (or control dCas9) vectors along with five guide RNAs (or a control empty vector) followed by brief selection of transfected cells under 5 μg/ml puromycin and subsequently analyzed by ChIP-qPCR.

FIG. 4B shows the analysis of the occupancy of Flag-tagged dCas9/dCas9^(p300 Core) protein at the K-M enhancer region by ChIP-qPCR in HEK293T cells 72 hours after transfection. An empty vector (lentiGuide puro vector) or a mixture of guide RNA constructs were transfected along with a dCas9 or dCas9^(p300 core) expression vector.

FIG. 4C Analysis of the occupancy of histone H3K27ac at the K-M enhancer region by ChIP-qPCR in HEK293T cells 72 hours after transfection. An empty vector (lentiGuide puro vector) or a mixture of guide RNA constructs were transfected along with a dCas9 or dCas9^(p300 core) expression vector.

FIG. 4D shows the MGMT expression in HEK293T cells was analyzed 72 hrs after co-transfection with either an empty vector or guide RNA construct along with a dCas9 or dCas9^(p300 core) expression vector. MGMT transcript level was first normalized to actin and subsequently calculated as fold change relative to double negative (Vector dCas9) control (*p<0.05, n=3 independent experiments).

FIG. 4E shows the analysis of the occupancy of Flag tagged dCas9/dCas9^(p300 core) protein at the K-M enhancer region by ChIP-qPCR in 5199 cells 72 hours after infection. 5199 cells were infected with a mixture of virus containing an empty vector (lentiGuide puro vector) or five guide RNA constructs and transfected with a dCas9 or dCas9^(p300 Core) construct.

FIG. 4F shows the analysis of the occupancy of histone H3K27ac at the K-M enhancer region by ChIP-qPCR in 5199 cells 72 hours after infection. 5199 cells were infected with a mixture of virus containing an empty vector (lentiGuide puro vector) or five guide RNA constructs and transfected with a dCas9 or dCas9^(p300 Core) construct.

FIG. 4G shows the MGMT expression in 5199 cells after targeting p300 core to the enhancer by CRISPR/dCas9. The experiments were performed as described from b-d (*p<0.05, n=3 independent experiments).

FIGS. 5A-5J shows that deletion of K-M enhancer reduces MGMT expression and increases sensitivity to TMZ.

FIG. 5A is an outline of deletion strategy using CRISPR/Cas9 system. Cells were infected with a mixture of two virus containing guide RNAs surrounding K-M enhancer locus. After puromycin selection, single clones were isolated and tested by PCR.

FIG. 5B shows that MGMT expression in SKMG3 parental cell, SKMG3 wild type clone and three K-M enhancer deleted clones were analyzed by quantitative RT-PCR. MGMT transcript level was first normalized to actin and subsequently calculated as fold change relative to SKMG3 parental line.

FIG. 5C shows that MGMT protein levels in SKMG3 parental cell, SKMG3 wild type clone and three K-M enhancer deleted clones were tested by Western blotting. α-tubulin was used as loading control.

FIG. 5D shows that SKMG3 parental line, wild type clone and K-M enhancer deleted clones were treated with indicated concentrations of temozolomide (0-1000 μM/L final concentration). Cell viability was determined using clonogenic assay.

FIG. 5E shows that SKMG3 parental line, wild type clone and K-M enhancer deleted clones were treated with 10 μM O6BG 1 h prior to temozolomide (0-1000 μM/L final concentration). Cell viability was determined using clonogenic assay.

FIG. 5F is a summary of TMZ IC₅₀ in SKMG3 clones with or without O6BG pre-treatment.

FIG. 5G shows that MGMT expression in SKMG3 parental cells and three Del1 deleted clones were analyzed by quantitative RT-PCR. MGMT transcript level was first normalized to actin and subsequently calculated as fold change relative to SKMG3 parental line.

FIG. 5H shows that SKMG3 parental cells and three Del1 deletedclones were treated with indicated concentrations of temozolomide (0-1000 μM/L final concentration). Cell viability was analyzed using clonogenic assay.

FIG. 5I shows that MGMT transcription levels in 3080 parental cells and 3080 Del1 deleted cells was analyzed by quantitative RT-PCR. MGMT transcript level was first normalized to actin and subsequently calculated as fold change relative to 3080 parental line.

FIG. 5J shows that 3080 parental cells and 3080 Del1 deleted cells were treated with different concentration of temozolomide (0-1000 μM/L final concentration). Cell viability was determined using neurosphere formation assay. (***p<0.001, ****p<0.0001, n=3 independent experiments)

FIGS. 6A-6E show that deletion of the K-M enhancer affects cell proliferation and Ki67 expression.

FIG. 6A is a schematic diagram showing the relative genomic location of MKI67 gene, the K-M enhancer and MGMT gene.

FIG. 6B shows that cell proliferation rate was analyzed by Incucyte. The cell confluency read out for each cell was normalized by the cell confluency acquired at the first time point.

FIG. 6C shows that MKI67 transcription levels in SKMG3 wild type clone and two enhancer deleted clones was analyzed by quantitative RT-PCR. Ki67 expression level was first normalized against actin and subsequently calculated as fold change relative to wild type clone.

FIG. 6D shows the growth rate of 3080 parental and 3080 Del1 cells injected flank tumors. Equal numbers of 3080 parental cells and 3080 Del1 cells were injected into the flank of mice (n=5/group). Tumor volume was used to represent growth rate.

FIG. 6E shows that MKI67 transcription levels in 3080 parental cells and 3080 Del1 deleted clone cells was analyzed by quantitative RT-PCR. Ki67 expression level was first normalized against actin and subsequently calculated as fold change relative to the parental line.

FIGS. 7A-7B show the average levels of H3K9me3 and H3K9ac in GBM xenografts.

FIG. 7A is an aggregate plot showing the average reads distribution for H3K9me3 ChIP-seq reads surrounding transcription start site.

FIG. 7B is an aggregate plot showing the average reads distribution for H3K9ac ChIP-seq reads surrounding transcription start site.

FIGS. 8A-8C show the reporter assay findings in 5199 and 3080 cells.

FIG. 8A is a schematic diagram showing the ten fragments (R1-R10) covering K-M enhancer region were cloned upstream of luciferase promoter.

FIG. 8B shows that effect of each fragment on transcription of luciferase was tested in 5199 cells. Firefly and Renilla luciferase activity were measured 36 h after transfection. Luciferase activity was normalized to the Renilla luciferase activity. Reported values were derived from 3 biological repeats. (*p<0.05, **p<0.01, ***p<0.001)

FIG. 8C shows that effect of each fragment on transcription of luciferase was tested in 3080 cells. Firefly and Renilla luciferase activity were measured 36 h after transfection. Luciferase activity was normalized to the Renilla luciferase activity. Reported values were derived from 3 biological repeats. (*p<0.05, **p<0.01, ***p<0.001)

FIGS. 9A-9C provide MGMT expression and enhancer activity profile for promoter hypermethylated PDX tumors.

FIG. 9A shows that MGMT transcription levels in eight MGMT promoter methylated PDX tumors were analyzed by quantitative RT-PCR. MGMT expression was normalized to actin.

FIG. 9B shows that H3K4me1 occupancy at the putative K-M enhancer region was analyzed in eight PDX tumors by ChIP-qPCR with primers described in FIGS. 2A-2D. Reported values were derived from three biological repeats. (*p<0.05, **p<0.01, ***p<0.001)

FIG. 9C shows that H3K27ac occupancy at the putative K-M enhancer region was analyzed in eight PDX tumors by ChIP-qPCR with primers described in FIGS. 2A-2D. Reported values were derived from three biological repeats. (*p<0.05, **p<0.01, ***p<0.001)

FIG. 10 shows CD45, Ki67, and MGMT triple immunofluorescence staining in primary and recurrent tumors from three GBM patients. Representative images showing CD45, MGMT and Ki67 co-localization in tissue sections from primary and recurrent tumor obtained from patients #1, #2, and #3. Multicolor immunofluorescence was performed using antibodies against CD45, MGMT and Ki67.

FIGS. 11A-11C provide PCR confirmation, Sanger sequencing and growth rate analysis for SKMG3 K-M enhancer deleted clones.

FIG. 11A is a gel image showing successful deletion of a 3.3 kb fragment in SKMG3 K-M enhancer deleted clones. One pair of primers spanning the sequence to be deleted was used for PCR assay.

FIG. 11B shows that a successful deletion of K-M enhancer in three SKMG3 clones was confirmed by Sanger sequencing. The location of the PAM sequence (underlined), the predicted cutting site (marked by arrow head) and the deletion patterns detected by Sanger sequencing of all K-M enhancer deletion clones were aligned on the top panel. The raw Sanger sequencing plot is displayed in the lower panel.

FIG. 11C provides images showing the clone size of SKMG3 parental cells, wild type clone and two deletion clones under DMSO treatment. The smaller clone size of enhancer deleted clones indicates impaired proliferation.

FIGS. 12A-12D provide PCR confirmation and MGMT expression for small deletion clones.

FIG. 12A is a gel image showing successful deletion of two 1.6 kb fragments in SKMG3 cells. One pair of primers spanning the sequence to be deleted was used for PCR assay. The regions to be deleted were indicated in top panel. Two pairs of primers, one spanning Del1 region another one spanning Del2 region, were used for PCR.

FIG. 12B shows that MGMT expression in SKMG3 parental line, wild type clone and Del2 deleted clones were analyzed by quantitative RT-PCR. MGMT expression level was normalized to actin and subsequently calculated as fold change relative to SKMG3 parental line.

FIG. 12C shows that the proliferation rate of SKMG3 parental cells and Del1 deleted SKMG3 clones were tested for proliferation using cell titer blue assay. The growth rate for each cell was normalized to Day 0 control.

FIG. 12D is a gel image showing successful deletion of Del1 region in 3080 cells. The PCR primers used in this assay was the same as described in FIGS. 11A-11C.

FIGS. 13A-13B show the genomic location of MKI67 gene and its expression in recurrent tumor from patient #1.

FIG. 13A is an IGV snapshot showing H3K4me1, H3K27ac, H3K4me3 and H3K36me3 read density on MKI67 gene, K-M enhancer and MGMT gene in 5199 and 3080 PDX tumors.

FIG. 13B shows that Ki67 expression level in each tumor was analyzed by immunofluorescence. The Ki67 signal (area×average intensity) from 100 individual nuclei was measured and one-way ANOVA analysis was used to calculate significance. (****p<0.0001, ns: not significant, n=100)

FIG. 14A shows that GBM cells (3080) treated with a p300/CBP inhibitor, CBP1, resulted in reduced levels of H3K27ac and reduced expression of MGMT.

FIG. 14B shows that GBM cells (3080) treated with a p300/CBP inhibitor, CPI-329, resulted in reduced levels of H3K27ac and reduced expression of MGMT.

FIG. 15A shows the result of RT-PCR analysis on the expression of MGMT in GBM cells treated with different concentrations of CBP1.

FIG. 15B shows the result of RT-PCR analysis on the expression of MGMT in GBM cells treated with different concentrations of CPI-329.

FIGS. 16A-16B show that p300 inhibition sensitized GBM cells to TMZ.

FIG. 16A shows the results of neurosphere formation assay (left) and colony formation assay (right), indicating that GBM cells treated with CBP1 resulted in increased sensitivity to TMZ.

FIG. 16B shows the results of neurosphere formation assay (left) and colony formation assay (right), indicating that GBM cells treated with CPI-329 resulted in increased sensitivity to TMZ.

DETAILED DESCRIPTION OF THE INVENTION

Temozolomide (TMZ) has been used for the treatment of glioblastoma (GBM) since last decade, but its treatment benefits are limited by acquired resistance, a process that remains incompletely understood. In the present invention, it is shown that a novel enhancer, located between the promoters of Ki67 and O₆-methylguanine-DNA-methyltransferase (MGMT) genes, is activated in TMZ resistant patient derived xenograft (PDX) lines as well as in recurrent tumor samples. Activation of the enhancer correlates with increased MGMT expression, a major known mechanism for TMZ resistance. It is also shown that forced activation of the enhancer in cell lines with low MGMT expression results in elevated MGMT expression. Deletion of this enhancer in cell lines with high MGMT expression leads to reduced levels of MGMT and Ki67, increased TMZ sensitivity and impaired proliferation. Together, the present invention is directed to a novel mechanism that regulates MGMT expression, confers TMZ resistance and potentially regulates tumor proliferation.

Accordingly, one aspect of the present invention is a method for treating or ameliorating the effects of a cancer in a subject. This method comprises administering to the subject a therapeutically effective amount of a first agent that modulates the activity of an enhancer and a therapeutically effective amount of a second agent that is used to treat the cancer.

In some embodiment of this aspect, the enhancer regulates the expression of O₆-methylguanine-DNA-methyltransferase (MGMT) and/or Ki67 in the subject. In certain embodiments, the enhancer is K-M enhancer that is situated between MKI67 and MGMT promoters and is 560 kb away from the MGMT promoter.

In some embodiment of this aspect, the first agent is selected from a Bromodomain inhibitor (BETi), a histone acetyltransferase inhibitor (HATi), and combinations thereof. Non-limiting examples of Bromodomain inhibitors include JQ1, I-BET151/762, PF-1, RVX-208, BMS-986158, OTX015, PLX-51107, GSK525762, INCB054329, TEN-010, GSK2820151, BAY 1238097, and combinations thereof. Non-limiting examples of histone acetyltransferase inhibitors include C646, NU-9056, PU141, EML425, L002, MB-3, CPTH2, Plumbagin, Embelin, EGCG, Curcumin, HAT inhibitor II, Garcinol, Anacardic acid, MG 149, Gossypol, CTK7A, Windorphen, LoCAM, TH1834, CTx-1, Lys-CoA, and combinations thereof. In certain embodiments, the histone acetyltransferase inhibitor (HATi) is a p300 HAT inhibitor. Non-limiting examples of p300 HAT inhibitors include curcumin (CAS #458-37-7), garcinol (CAS #78824-30-3), anacardic acid (CAS #16611-84-0), C646 (CAS #328968-36-1), Demethoxy curcumin (CAS #22608-11-3), 5-Chloro-2-(4-nitrophenyl)-3(2H)-isothiazolone (CAS #748777-47-1), Histone Acetyltransferase Inhibitor II (CAS #932749-62-7), L002 (CAS #321695-57-2), CBP1, CPI-329. In some embodiments, the p300 HAT inhibitor is selected from CBP1, CPI-329, and combinations thereof.

In some embodiments of this aspect, the second agent is temozolomide (TMZ).

In some embodiments of this aspect, the subject is TMZ-resistant.

In some embodiments of this aspect, the cancer is selected from breast cancer, lung cancer, and brain tumor. In certain embodiments, the brain tumor is gliobastoma (GBM).

As used herein, the terms “treat,” “treating,” “treatment” and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present invention may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.

As used herein, a “subject” is a mammal, preferably, a human.

In some embodiments of this aspect, this method further comprises administering to the subject a therapeutically effective amount of a histone deaceylase inhibitor (HDACi). Non-limiting examples of histone deaceylase inhibitors include Trichostatin A, SAHA, Belinostat, Panabiostat, Givinostat, Resminostat, Abexinostat, Quisinostat, Rocilinostat, Practinostat, CHR-3996, Valproic acid, Butyric acid, Phenylbutyric acid, Entinostat, Tacedinaline, 4SC202, Mocetinostat, Rom idepsin, Nicotinamide, Sirtinol, Cambinol, EX-527, and combinations thereof.

Another aspect of the present invention is a method for delaying the emergence of temozolomide (TMZ) resistance and/or increasing the TMZ sensitivity in a subject. This method comprises administering to the subject a therapeutically effective amount of an agent that modulates the activity of an enhancer.

In some embodiments of this aspect, the enhancer regulates the expression of O₆-methylguanine-DNA-methyltransferase (MGMT) in the subject. In certain embodiments, the enhancer is K-M enhancer that is situated between MKI67 and MGMT promoters and is 560 kb away from the MGMT promoter.

In some embodiment of this aspect, the agent is selected from a Bromodomain inhibitor (BETi), a histone acetyltransferase inhibitor (HATi), and combinations thereof. Non-limiting examples of Bromodomain inhibitors include JQ1, I-BET151/762, PF-1, RVX-208, BMS-986158, OTX015, PLX-51107, GSK525762, INCB054329, TEN-010, GSK2820151, BAY 1238097, and combinations thereof. Non-limiting examples of histone acetyltransferase inhibitors include C646, NU-9056, PU141, EML425, L002, MB-3, CPTH2, Plumbagin, Embelin, EGCG, Curcumin, HAT inhibitor II, Garcinol, Anacardic acid, MG 149, Gossypol, CTK7A, Windorphen, LoCAM, TH1834, CTx-1, Lys-CoA, and combinations thereof. In certain embodiments, the histone acetyltransferase inhibitor (HATi) is a p300 HAT inhibitor.

Another aspect of the present invention is a method for reducing tumor aggressiveness in a subject. This method comprises administering to the subject a therapeutically effective amount of an agent that modulates the activity of an enhancer.

In some embodiments of this aspect, the enhancer regulates the expression of Ki67 in the subject. In certain embodiments, the enhancer is K-M enhancer that is situated between MKI67 and MGMT promoters and is 560 kb away from the MGMT promoter.

In some embodiment of this aspect, the agent is selected from a Bromodomain inhibitor (BETi), a histone acetyltransferase inhibitor (HATi), and combinations thereof. Non-limiting examples of Bromodomain inhibitors include JQ1, I-BET151/762, PF-1, RVX-208, BMS-986158, OTX015, PLX-51107, GSK525762, INCB054329, TEN-010, GSK2820151, BAY 1238097, and combinations thereof. Non-limiting examples of histone acetyltransferase inhibitors include C646, NU-9056, PU141, EML425, L002, MB-3, CPTH2, Plumbagin, Embelin, EGCG, Curcumin, HAT inhibitor II, Garcinol, Anacardic acid, MG 149, Gossypol, CTK7A, Windorphen, LoCAM, TH1834, CTx-1, Lys-CoA, and combinations thereof. In certain embodiments, the histone acetyltransferase inhibitor (HATi) is a p300 HAT inhibitor.

Yet another aspect of the present invention is a pharmaceutical composition. This pharmaceutical composition comprises 1) a therapeutically effective amount of a first agent that modulates the activity of an enhancer in a subject suffering from a cancer and 2) a therapeutically effective amount of a second agent that is used to treat the cancer.

In some embodiment of this aspect, the enhancer regulates the expression of O₆-methylguanine-DNA-methyltransferase (MGMT) and/or Ki67 in the subject. In certain embodiments, the enhancer is K-M enhancer that is situated between MKI67 and MGMT promoters and is 560 kb away from the MGMT promoter.

In some embodiment of this aspect, the first agent is selected from a Bromodomain inhibitor (BETi), a histone acetyltransferase inhibitor (HATi), and combinations thereof. Non-limiting examples of Bromodomain inhibitors include JQ1, I-BET151/762, PF-1, RVX-208, BMS-986158, OTX015, PLX-51107, GSK525762, INCB054329, TEN-010, GSK2820151, BAY 1238097, and combinations thereof. Non-limiting examples of histone acetyltransferase inhibitors include C646, NU-9056, PU141, EML425, L002, MB-3, CPTH2, Plumbagin, Embelin, EGCG, Curcumin, HAT inhibitor II, Garcinol, Anacardic acid, MG 149, Gossypol, CTK7A, Windorphen, LoCAM, TH1834, CTx-1, Lys-CoA, and combinations thereof. In certain embodiments, the histone acetyltransferase inhibitor (HATi) is a p300 HAT inhibitor.

In some embodiments of this aspect, the second agent is temozolomide (TMZ).

In some embodiments of this aspect, the subject is TMZ-resistant.

In some embodiments of this aspect, the cancer is selected from breast cancer, lung cancer, and brain tumor. In certain embodiments, the brain tumor is gliobastoma (GBM).

In some embodiments of this aspect, this method further comprises administering to the subject a therapeutically effective amount of a histone deaceylase inhibitor (HDACi). Non-limiting examples of histone deaceylase inhibitors include Trichostatin A, SAHA, Belinostat, Panabiostat, Givinostat, Resminostat, Abexinostat, Quisinostat, Rocilinostat, Practinostat, CHR-3996, Valproic acid, Butyric acid, Phenylbutyric acid, Entinostat, Tacedinaline, 4SC202, Mocetinostat, Rom idepsin, Nicotinamide, Sirtinol, Cambinol, EX-527, and combinations thereof.

A further aspect of the present invention is a kit. This kit comprises the aforementioned pharmaceutical composition, and instructions of use.

Still another aspect of the present invention is a method for treating or ameliorating the effects of gliobastoma (GBM) in a subject that is resistant to temozolomide (TMZ). This method comprises administering to the subject a therapeutically effective amount of a p300 HAT inhibitor or a Bromodomain inhibitor (BETi) and a therapeutically effective amount of TMZ.

An additional aspect of the present invention is a method of modulating proliferation of brain tumor cells with or without activation of a MGMT enhancer. This method comprises contacting the cells with an effective amount of a composition selected from a HAT inhibitor, a p300 inhibitor, a bromodomain inhibitor, and combinations thereof.

The invention is further illustrated by the following examples, which are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters, which can be changed or modified to yield essentially the same results.

EXAMPLES Example 1 Methods and Materials Cell Culture

GBM cell line SKMG3 (provided by Dr. David James) and HEK293T cell line procured from ATCC were maintained in DMEM (CORNING, 10-013-CV) supplemented with 10% fetal bovine serum (Millipore Sigma, TMS-013-B) and 1% Penicillin-Streptomycin (CORNING, 30-001-CI).

GBM xenograft sublines GBM12 5199 and GBM12 3080 were developed from GBM12 patient derived xenograft and were propagated in the form of subcutaneous xenografts in athymic nude mice as previously described 26; primary cells from xenograft tissues were cultured in StemPro NSC media and supplements (ThermoFisher, A1050901) as previously described (Kitange et al. 2012).

Xenograft Tumors

Frozen tumor tissues from xenografts established from primary GBM (GBM43, GBM59, GBM61, GBM115 and GBM122) and those from recurrent GBM (G46, G64 and G102) were developed in our lab as previously described (Kitange et al. 2017).

Paired Patient Samples for ChIP-qPCR and Immunofluorescence Analysis

Studies involving patient samples were approved by the Mayo Clinic Institutional Review Board (IRB number 09-003015). Paired (primary and recurrent) GBM frozen tissue samples collected from consented patients, who had hypermethylated MGMT promoter at primary diagnoses and received standard TMZ treatment, were obtained from Mayo Clinic Neuro-Oncology tissue bank. A total of 3-pairs of frozen tumors with high tumor cellularity (>80%), large sample size (>30 mg) and hypermethylated MGMT promoter were selected. Tissues were cryosectioned and subjected to concomitant immunofluorescence and ChIP-qPCR analyses. Tumor samples for ChIP-qPCR were collected by scratching the tumor dense areas from the tissue sections, taking corresponding H&E stained slides as reference.

Antibodies

Antibodies against MGMT (R&D, AF3794) and anti-α-Tubulin (Developmental Studies Hybridoma bank, 12G10) were used in western blotting analysis.

Antibodies against H3K4me1 (Abcam, Ab8895), H3K27ac (Abcam, Ab4729), H3K9ac (Abcam, Ab4441), H3K4me3 (Abcam, Ab8580), H3K9me3 (Active Motif, 39161), H3K36me3 (Active Motif, Cat #61101) and Flag (Sigma-Aldrich, 11583816001) were used for chromatin immunoprecipitation assay.

Monoclonal antibodies against CD45 (Cell Signaling, Cat #13917), MGMT (Millipore, Cat #MAB16200), Ki67 (Thermo Fisher, Cat #14-5698-82) and conjugated Alexa Fluor 488 labeled goat anti rabbit IgG (Jackson Immuno Research, Cat #111-545-144), Alexa Fluor 594 labeled goat anti mouse IgG (Life Technologies, Cat #A11032) and Cy5 labeled goat anti rat IgG (Jackson Immuno Research, Cat #112-175-167) were used for immunofluorescence.

Western Blot Assay

Western blots were performed as previously described (Fang et al. 2016). Primary antibodies were used under 1:1000 dilution.

RNA Isolation Reverse Transcription and Real-Time PCR

RNA was extracted with RNeasy plus kit (Qiagen, #74134) according to the manufacturer's instructions. Reverse transcription of mRNA was performed using SuperScript™ III Reverse Transcriptase (Invitrogen, 18080-085). For real time PCR analysis, 1 μl of cDNA (25 ng of starting RNA) was amplified per reaction using the iTaq Universal SYBR Green Supermix (Bio-Rad, 172-5124) and the Bio-Rad CFX qPCR system as previously described (Fang et al. 2016). Primers for real time qPCR analysis were listed in Table 1.

RNA-Seq

RNA sequencing was performed as previously described (Kitange et al. 2016). Briefly, total RNA was extracted as described above. The RNA quality was further evaluated using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, Calif.). The Illumina TrueSeq RNA Sample preparation Kit v.4.1 (Illumina Inc., San Diego, Calif.) was used to prepare cDNA libraries from 2 μg of total RNA for RNA-seq. Individual barcoded libraries were analyzed using Agilent 2100 Bioanalyzer (Agilent technologies). Sequencing was carried out on an Illumina HiSeq 2000 machine (Illumina) at Mayo Clinic Medical Genomic Facility.

Ms-PCR

DNA was extracted from frozen tissues or cells using Blood & Cell Culture DNA Mini Kit (Qiagen, #13323). Isolated genomic DNA was bisulfite-treated with EZ DNA methylation Gold kit (Zymo Research, #11-335B). The modified DNA was quantified by PCR. MS-M-F/R primers were used to PCR methylated MGMT promoter, MS-U-F/R primers were used to PCR unmethylated MGMT promoter. The sequences for PCR primers were listed in Table 1.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation assay was performed as described (Fang et al. 2016). 1×10⁶ cells or 15 mg homogenized frozen tissues were fixed with 1% paraformaldehyde at room temperature for 10 min, quenched with 0.125 M glycine and lysed for 10 min on ice. The lysate was digested with MNase (NEB, Cat #M0247S) at 2000 gel unit/ml final concentration at 37° C. for 20 min and sonicated 15 cycles (30 s on, 30 s off) under high power using a Diagenode Bioruptor. Crosslinked DNA was immunoprecipitated with 2 μg antibody at 4° C. overnight, pulled down by protein G beads, washed, reverse crosslinked and purified for qPCR for high throughput sequencing analysis.

For ChIP-qPCR analysis, both input and immunoprecipitated DNA were quantified by real-time PCR with primers listed in Table 1. DNA quantity for each ChIP sample was normalized against input DNA.

For ChIP-seq samples, after DNA purification ChIP-seq DNA libraries were prepared with the Ovation Ultralow DR Multiplex system (NuGEN). The DNA libraries were sequenced using the 51 bp paired-end sequencing method by an IIlumina Hi-seq 2000.

ChIP-Seq Analysis

Raw reads from IIlumina Hi-seq 2000 were aligned to the human genome (hg19) using Bowtie2 software with default parameters. Only uniquely mapping reads were used for secondary analysis. The ChIP-seq peaks were identified by MACS2 using the default calling parameter; and the p value of cutoff was set to 0.001. Genome-wide read coverage was calculated by BEDTools and in-house Perl programs, and visualized using Integrative Genomics Viewer as previously described (Zhang et al. 2017). The reads density scan was performed by in-house Perl programs using the traditional normalization method: Reads per Kilobase per Million Mapped Reads (RPKM).

For cluster analysis, firstly genomic regions with high H3K4me1 and low H3K4me3 occupancy in 5199 and 3080 cells were selected. H3K4me1 peaks were merged if their distance is less than 500 bp. The

$\log_{2}\frac{H\; 3K\; 4{me}\; 1(3080)}{H\; 3K\; 4{me}\; 1(5199)}\mspace{14mu} {and}\mspace{14mu} \log_{2}\frac{H\; 3K\; 27{{ac}(3080)}}{H\; 3K\; 27{{ac}(5199)}}$

on those merged peaks were used for unsupervised k-means cluster analysis.

Pathway Analysis

The Group-1 genes identified from cluster analysis were imported into Ingenuity pathway analysis (IPA) program. A list of affected pathways was calculated based on default setting.

Reporter Assay

DNA fragments tested in a reporter assay are named as reporter fragments (R1 to R10). Those fragments were inserted upstream of SV40 promoter and Firefly luciferase in a pGL3 promoter vector. For each transfection reaction, 100 ng control plasmid expressing Renilla luciferase and 300 ng Firefly luciferase construct were co-transfected into 2×10⁵ cells in a 24 well plate well. After 24 h, luciferase activities were measured by the Dual-Luciferase Reporter Assay System (Promega, E1910).

Chromatin Conformation Capture Assay

The chromatin conformation capture assay was performed as described (Hagege et al. 2007). Briefly, cells were crosslinked with 1% formaldehyde at room temperature for 10 min, quenched with 0.125 M glycine, lysed, and treated with 600 U HindIII (NEB, #R3104) at 37° C. overnight followed by a 4 h ligation with T4 enzyme (NEB, M0202L) at 16° C. Ligated products were quantified in triplicate by TaqMan real-time PCR. Probes and primers (listed in Table 1) were designed by using primer blast provided by NCBI. Control 3C template was generated by using two bacterial artificial chromosomes (BACs), 656G14 and 1125P18, which together encompass putative K-M enhancer and MGMT promoter regions. Equimolar of the two BACs were digested with HindIII and ligated. The ligation product from BAC control was used for normalization. The relative interaction frequency was calculated as: 2^(Ct(BAC)-Ct(3C))

Immunofluorescence

OCT embedded patient tumor tissues were sectioned at 5 micron thickness. Slides were fixed by 4% paraformaldehyde at 4° C. for 10 minutes, penetrated with PBS plus 0.25% Triton X-100 for 10 min and pretreated with steam TBS antigen retrieval buffer at pH 9.0 for 60 minutes. One hour primary antibody incubation (1:100 dilution) was performed at room temperature followed by one hour secondary antibody incubation (1:200 dilution). Slides were rinsed, dehydrated and mounted with Prolong Gold antifade mounting media with DAPI (Invitrogen, Cat #P36935) and analyzed by confocal microscopy (LSM 780; 63× objective lenses). MGMT and Ki67 were considered positive when uniform staining was detected in cell nuclei. CD45 was considered positive when cytoplasmic staining was detected.

The signal intensity for each cell was quantified in ImageJ program. The area of each nucleus was determined by DAPI staining. The total signal for each nucleus was calculated as: signal=area×average signal intensity. The total signal for 100 nuclei from each slides were quantified and plotted.

The investigator was blinded to sample allocation during immunofluorescence image collection and counting.

Guide RNA Design and Cloning

All guide RNAs were designed by using MIT CRISPR Design website (http://crispr.mit.edu). To minimize potential off-target effects of guide RNA, only high score guide RNAs (score >85) were used. Guide RNA sequences are listed in Table 2.

Guide RNAs used in CRISPR/dCas9 system were cloned into lentiGuide puro vector (Addgene, Plasmid #52963) by using a published protocol (Sanjana et al. 2014). Guide RNAs used in CRISPR/Cas9 were cloned into lentiCRISPRv2 vector using the same protocol.

Enhancer Activation by CRISPR/dCas9^(p300 Core) System

Guide RNAs were cloned into lentiGuide puro vector (Addgene, Plasmid #52963). 5199 cells were infected with a mixture of virus containing five guide RNAs while HEK293T cells were co-transfected with a pooled guide RNA containing five guide RNA constructs. Both 5199 cell and HEK293T cells are transfected with a pcDNA-dCas9-p300 (or control dCas9) Core plasmid (Addgene, Plasmid #61357). Puromycin selection was performed 24 h post transfection. Targeting of dCas9^(p300 Core) protein was confirmed by Flag ChIP-qPCR assay at 72 h post transfection. Enhancer activity and MGMT transcript were assessed by H3K27ac ChIP-qPCR and RT-PCR respectively. Primers used for ChIP-qPCR and RT-PCR assay are listed in Table 1.

CRISPR/Cas9-Mediated Genomic Deletion

Guide RNAs were cloned into lentiCRISPR v2 vector (Addgene, Plasmid #52961). Lentivirus for guide RNAs were produced as previously described (Zhang et al. 2017). For genomic deletion model, SKMG3 cells were infected with equal amount of two lentivirus carrying guide RNAs flanking the region to be deleted, followed by clonal selection under puromycin and clone expansion. Paired guide RNAs g1/g2 were used to generate larger deletion, while paired guide RNAs g1/g3 and g2/g3 were used to generate the first smaller deletion and the second smaller deletion. PCR amplification was used for genotypic characterization of putative deletion clones. The sequence of those primers is listed in Table 1. The PCR products of positive clones with homozygous deletion were validated by Sanger sequencing.

Clonogenic Assay

Clonogenic assays were performed to assess the effect of disrupted enhancer on TMZ sensitivity. Briefly, SKMG3 parental cells and enhancer deletion clones were plated in 6 wells plates (250 cells/well), treated with graded concentration of TMZ in presence or absence of O6BG and were cultured for 2 weeks. Colonies were fixed and stained with crystal violet (0.005% (w/v) Crystal violet, 25% (v/v) Methanol). Colonies with >50 cells were manually counted, IC₅₀ calculated by GraphPad Prism 7 using multiparametric nonlinear regression model.

Neurosphere Assay

Neurosphere assay was performed as previously described (Kitange et al. 2012). In brief, primary cells suspended in StemPro NSC media were plated in triplicate in 96-well plates (500 cells per well) and treated with graded concentration of TMZ (0-1000 μM/L final concentration). Intact neurospheres containing more than 50 cells were counted after 15 days. Cell viability was calculated relative to DMSO control. IC₅₀ was calculated by GraphPad Prism 7 7 using multiparametric nonlinear regression model.

Cell Proliferation Assay

IncuCyte machine was used to measure cell proliferation rate of SKMG3 wild type clone and K-M enhancer deleted clones. Cells were plated in 96 well plates in triplicate at 500 cells per well density. The percentage of confluency for each well was measured for 6 days. The confluency data was used to calculate proliferation rate as fold changes compare to Day 0.

CellTiter-Blue® Cell Viability Assay kit (CTB) was used to measure cell viability for SKMG3 and SKMG3-Del1 clones. Cells were plated in 96-well plates in triplicate at 1,000 cells per well density. Cell number was measured every other day according to kit manufacturer's instruction. Fluorescence signal was read by GloMax®-Muti Microplate Multimode Reader with excitation at 560 nm and emission at 590 nm.

Flank Tumor Injection

1×10⁶ cells were injected into the flank of athymic mice. Mice were euthanized and tumors harvested when the tumor size exceeded 2,000 mm³.

Statistical Analysis

ChIP-qPCR analysis for patient samples was not replicated due to limited sample size. All other experiments were done with three biological replicates.

A two-tailed Student's t test was used to establish statistical significance between control and testing group for all comparison between two data sets. One way ANOVA was used for MGMT and Ki67 signal intensity comparison analysis.

TABLE 1 Primers and TaqMan probe sequences used for MS-PCR and qPCR. Primer name Sequence(5′-3′) Assay Actin-F AGAGCTACGAGCTGCC RT-PCR TGAC (SEQ ID NO: 1) Actin-R AGCACTGTGTTGGCG RT-PCR TACAG (SEQ ID NO: 2) MGMT-F GCTGCGGTTCTCGGAG RT-PCR GTC (SEQ ID NO: 3) MGMT-R CTGCCAGGGCTGCTAA RT-PCR TTGC (SEQ ID NO: 4) MS-M-F TTTCGACGTTCGTAG MS-PCR GTTTTCGC (SEQ ID NO: 5) MS-M-R GCACTCTTCCGAAAAC MS-PCR GAAACG (SEQ ID NO: 6) MS-U-F TTTGTGTTTTGATGTT  MS-PCR TTGTAGGTTTTTGT (SEQ ID NO: 7) MS-U-R AACTCCACACTCTT MS-PCR CCAAAAACAAAACA (SEQ ID NO: 8) PE1-F TACCAGGAATGATGTGG ChIP-qPCR GGTTA (SEQ ID NO: 9) PE1-R GGCATCTTCCCCTTCA ChIP-qPCR CAT  (SEQ ID NO: 10) PE2-F TGAACATGGCTTTTCA ChIP-qPCR AAGGA (SEQ ID NO: 11) PE2-R CAGCATTCCATGGTTG ChIP-qPCR ATGT (SEQ ID NO: 12) PE3-F CCATGCAGGCATTTCT ChIP-qPCR ACCT (SEQ ID NO: 13) PE3-R GAGCTCGCAGGAGCAA ChIP-qPCR CTAC (SEQ ID NO: 14) PE + 5kb − F TGCAAAATTTATTTG  ChIP-qPCR GGAGAAAA (SEQ ID NO: 15) PE + 5kb − R AGCTGCCATATAGAA ChIP-qPCR AATTGGTC (SEQ ID NO: 16) Digestion  CCTTTTGATAAATGCT 3C test TTCTGGA primer (SEQ ID NO: 17) Anchor primer ATAGCCTAATCTGCCA 3C AACGA (SEQ ID NO: 18) TaqMan Probe FAM-CATGTCTT 3C AAGCCACATACA TTTGGTTCCT- TAMRA (SEQ ID NO: 19) 3C F1 primer CCTTTCAGGCTTTATG 3C GCACC (SEQ ID NO: 20) 3C F2 primer TTGAATCATCTCTGGT 3C TCTGTTT (SEQ ID NO: 21) 3C F3 primer TCATCTTCAAATACTA 3C CTATGCCACTT (SEQ ID NO: 22) 3C F4 primer TCAGTCTTTGCATCCC 3C CTTTT (SEQ ID NO: 23) 3C F5 primer GCCTTCTCACCCAGT  3C ACACAA (SEQ ID NO: 24) 3C F6 primer GGCTGGCCTTGTACTC 3C TCT (SEQ ID NO: 25) MGMT TSS F GGGAGAGAGAGAGAAA 3C GATCTCC (SEQ ID NO: 26) MGMT TSS R GAGGCAGGGATGTCCA 3C TAAA (SEQ ID NO: 27) P1 AAAATCACAGCACCGC K-M deletion ACAG confirmation-F (SEQ ID NO: 28) P2 TTGCAGTCTGGCTTCC K-M deletion TCAA confirmation-R (SEQ ID NO: 29) Del1-P1 AAAATCACAGCACCGC Del1 deletion ACAG confirmation-F (SEQ ID NO: 30) Del1-P2 CTCGCAGGAGCAACTA Del1 deletion CT confirmation-R (SEQ ID NO: 31) Del2-P1 TGACAAAGAAACCCAT Del2 deletion CAGT  confirmation-F (SEQ ID NO: 32) Del2-P2 TTTGACCTATGGGAGA Del2 deletion AGTAGA confirmation-R (SEQ ID NO: 33)

TABLE 2 Guide RNA sequences for CRISPR-dCas9 based P300 targeting assay and CRISPR-Cas9 based genomic deletion assays. guide RNA names Sequence Used for g1 GCAGTGGCTTTCGCATAGTC Deletion (SEQ ID NO: 34) g2 GACAGGTGACTCTCGAAGAC Deletion (SEQ ID NO: 35) g3 TTACAAGTACTTACTCGCAT Deletion/ (SEQ ID NO: 36) Targeting T1 CAGCTCCTAGCTGAGTGTAA Targeting (SEQ ID NO: 37) T2 CTAACGTGCTCAGAAGATGG Targeting (SEQ ID NO: 38) T3 CCGATTTCTAAGTGCACAAA Targeting (SEQ ID NO: 39) T4 GACTCCGTCGTCCTGCCACG Targeting (SEQ ID NO: 40)

Example 2 Altered Histone Modifications at Enhancers in a TMZ Resistant GBM Xenograft Line

To investigate the genetic and epigenetic changes that occur during tumor recurrence, we used a patient-derived xenograft (PDX) model previously described (Kitange et al. 2012). The GBM12 xenograft line derived from a newly diagnosed MGMT hypermethylated tumor was used to generate TMZ resistant sublines. For this, multiple mice with flank tumors generated from GBM12 were treated with 3 cycles of TMZ or placebo. Two tumor sub-lines, a TMZ sensitive tumor from the placebo group named GBM12-5199 (5199) and a TMZ resistant tumor from the TMZ treatment group named GBM12-3080 (3080) were obtained (FIG. 1A). Similar to the original hypermethylated GBM12 tumor, the placebo-treated 5199 line had low MGMT protein expression and was highly susceptible to TMZ. In contrast, the TMZ-resistant 3080 line had robust MGMT expression despite of the presence of MGMT promoter methylation (FIGS. 1B-1C). These results suggest that other mechanisms were driving MGMT expression even in the presence of the MGMT promoter methylation.

In addition to DNA methylation, histone modifications also play an important role in the regulation of gene expression (Kitange et al. 2012; Allis et al. 2016). To test whether changes in histone modifications were associated with elevated MGMT expression in 3080, we analyzed histone modifications for active enhancers (H3K4me1 and H3K27ac), promoters (H3K4me3 and H3K9ac), or gene bodies (H3K36me3) and repressed mark (H3K9me3) in 5199 and 3080 sublines using Chromatin Immunoprecipitation coupled with next-generation sequencing (ChIP-seq) (FIGS. 1D-1G and FIGS. 7A-7B). We found that the global levels of H3K4me3 and H3K9ac at promoters and H3K36me3 at gene bodies were similar between 5199 and 3080. Interestingly the levels of H3K4me1 and H3K27ac at the enhancer regions were globally reduced in the 3080 line compared to the 5199 line. Moreover, the level of H3K9me3 was increased at the transcription starting sites (TSS) in the TMZ-resistant 3080 compared to the TMZ-sensitive 5199 (FIGS. 7A-7B). We noted that a previous reports showing that H3K9me2/3 increases globally in GBM cells upon TMZ treatment (Braig et al. 2005). These results suggest that histone modifications are susceptible to alterations during the acquisition of TMZ resistance.

Enhancer activation, which is characterized by increased H3K27ac at enhancers regions, leads to elevated transcription of nearby genes. To classify enhancers, putative enhancers with at least one enhancer mark altered in the recurrent 3080 line were used for unsupervised clustering analysis based on the changes of H3K4me1 and H3K27ac levels and the correlations with the expression change of genes close to each putative enhancer (FIG. 1H). In comparison to 5199 line, we observed that most enhancers exhibited reduced levels of H3K27ac and H3K4me1 in 3080 line, consistent with our previous observation that these two marks were reduced globally in 3080 compared to 5199 line. A small group of enhancers (1141 Group-1 enhancers) exhibited increased H3K27ac and H3K4me1 in 3080 line, suggesting that this group of enhancers is activated in the TMZ resistant line. Moreover, the activation of this group of enhancers correlated with increased expression of nearby genes. Pathway analysis of this group of genes indicated that genes that are involved in gliomagenesis and cancer drug resistance are enriched in this group of genes (FIG. 1I). These results suggest that a subgroup of enhancers was activated in the 3080 line and may contribute to TMZ resistance.

Example 3 MGMT is Regulated by a Novel Enhancer

Interestingly, MGMT, a key driver of TMZ resistance (Gerson et al. 2004), was one of the top 10 genes in the Group-1 gene list (Table 3). We therefore inspected H3K4me1, H3K27ac, H3K4me3 and H3K36me3 ChIP-seq peaks close to the MGMT gene locus (FIG. 2A). In line with increased MGMT transcription, H3K36me3 within the gene body and H3K4me3 in the promoter region were enriched in the 3080 line compared to 5199 line. Moreover, we also detected an increase in H3K4me1 and H3K27ac enrichment in the 3080 line compared to 5199 line at a region 560 kb away from the MGMT promoter, suggesting that this region may be a putative enhancer that can activate MGMT expression. Because this putative enhancer was localized in an intergenic region between MKI67 gene and MGMT gene, we named this putative enhancer K-M enhancer. By analyzing immunoprecipitated chromatin DNA using four pairs of primers, including three pairs spanning the putative K-M enhancer (PE1-3) region and a control region 5 kb away from putative enhancer region (PE+5 kb), with quantitative PCR (ChIP-qPCR), we confirmed that the levels of H3K27ac was significantly higher in the 3080 line than the 5199 line (FIG. 2B), whereas an equivalent level of H3K4me1 within the putative enhancer was detected between these two lines. The discrepancy in H3K4me1 at the putative enhancer detected by ChIP-qPCR and ChIP-seq may be due to the fact that H3K4me1 can be detected in the distal region. Alternatively, this putative enhancer could be in primed state, characterized by the presence of H3K4me1 and low levels of H3K27ac in the 5199 line. Collectively, these results suggest that a putative enhancer is activated in the TMZ resistant 3080 line and potentially drives MGMT expression.

Enhancers are regulatory elements that can promote gene expression. Therefore, we first tested whether this novel enhancer region can enhance transcription of a luciferase reporter gene. We cloned 10 DNA fragments (R1 to R10), each 1-2 kb in size, spanning the 13.5 kb H3K27ac peak region, in front of an SV40 promoter-driven firefly luciferase reporter. Each reporter construct was co-transfected with a pRL Renilla luciferase control reporter construct, which constitutively expresses Renilla luciferase to allow for normalization of transfection efficiency. While R1, R2, R6, R7, R9 and R10 significantly stimulated transcription compared to pGL3 vector control (FIGS. 8A-8C), only the 1.5 kb R7 fragment, localized in the second H3K27ac peak region, exhibited significantly higher activity in the 3080 line compared to 5199 (FIG. 2C). These results suggest that this region can enhance gene transcription and the R7 region may act as an MGMT enhancer, whereas other regions may serve as enhancer for other genes such as Ki67.

Enhancers typically contact with their cognate gene promoters through long-range interactions (Sur et al. 2016; Hnisz et al. 2013; Herz et al. 2014). We next tested whether the putative enhancer interacts with the MGMT promoter using the Chromatin Conformation Capture (3C) assay (FIG. 2D). In 3080 cells, a strong interaction between K-M enhancer and MGMT promoter was identified. The fourth test fragment (F4), which overlaps with the R7 region tested in the reporter assay, displayed significantly higher interaction frequency with the MGMT promoter compared to the neighboring DNA fragments. Moreover, the interaction frequency between the F4 fragment and the MGMT promoter in the 5199 line, which lacks MGMT expression, was significantly lower than the 3080 line, supporting the idea that the putative K-M enhancer region specifically interacts with the MGMT promoter in the 3080 line. Therefore, the 1.5 kb region located 560 kb away from the MGMT promoter has characteristics of an active enhancer (surrounding by nucleosomes with high H3K27ac, transcription enhancement, and interaction with promoter). The differential activity between the placebo 5199 line and the TMZ resistant 3080 line further underscored the idea that this novel enhancer was specifically activated to stimulate MGMT transcription, even in the presence of MGMT promoter methylation in 3080 line.

TABLE 3 Top 10 most strongly altered genes in Group-1. Rank Gene name Full name  1 MGMT O-6-Methylguanine-DNA Methyltransferase  2 ST18 Human suppression of tumorigenicity 18  3 WDFY4 WD repeat-and FYVE domain-containing protein 4  4 C17orf50 Chromosome 17 Open Reading Frame 50  5 WBSCR17 Williams-Beuren Syndrome Chromosome Region 17  6 ACTL8 Actin Like 8  7 MAP3K21 Mitogen-activated protein kinase kinase kinase 21  8 SORCS3 Sortilin Related VPS10 Domain Containing Receptor 3  9 BHLHA9 Basic Helix-Loop-Helix Family Member A9 10 TTR Transthyretin Strongly elevated genes within the 1141 genes in Group-1 are listed in the table. Genes were sorted from top to bottom based on the ratio of its expression in 3080 line divided by its expression in 5199 line.

Example 4 The Enhancer is Active in a Fraction of PDX Lines and Primary Tumor Samples

In addition to the 3080 line, we have detected MGMT expression in four MGMT promoter methylated xenograft lines previously (data not shown). To investigate whether the enhancer was activated in any of these MGMT expressing, promoter methylated PDX lines, we analyzed H3K4me1 and H3K27ac using ChIP-PCR in eight MGMT promoter methylated PDX lines including 4 PDX lines expressing MGMT (GBM43, GBM64, GBM115 and GBM122) and 4 lines without MGMT expression (GBM46, GBM59, GBM61 and GBM102) (FIG. 9A). H3K4me1 ChIP-qPCR analysis showed that 6 PDX lines (GBM43, GBM64, GBM115, GBM46, GBM61 and GBM102) had high levels of H3K4me1 at the enhancer region compared to a fragment 5 kb away (FIG. 9B). H3K27ac ChIP-qPCR analysis indicated that of the 6 samples with H3K4me1, three samples (GBM64, GBM115, and GBM46) had higher levels of H3K27ac at the K-M enhancer region compared to control locus (FIG. 9C), suggesting that the K-M enhancer is activated in these three lines. The GBM46 line is a MGMT low-expressing line from a recurrent tumor, whereas the two MGMT expressed lines, GBM115 and GBM64 are from primary and recurrent tumor, respectively, and have high levels of MGMT. Although the levels of H3K4me1 and H3K27ac enrichment at the K-M enhancer locus do not correlate with MGMT expression in GBM46 line, activation of the enhancers in two other MGMT promoter methylated PDZ lines correlates with high levels of MGMT expression, suggesting that enhancer activation is one explanation for the discordance between MGMT promoter methylation and gene expression.

To determine whether enhancer activation can also be detected in primary tissues, we chose to analyze H3K4me1 and H3K27ac at the enhancer locus in paired primary and recurrent GBM tumors with a methylated MGMT promoter. Sixty four paired frozen patient tumor samples at Mayo Clinic were identified and further filtered based on presence of MGMT promoter hypermethylation, TMZ treatment prior to resection of recurrent disease and the suitable tumor cell density and tissue quality to yield three pairs for subsequent ChIP-qPCR analysis (Table 4). Compared to the primary tumor, both H3K4me1 and H3K27ac levels increased in the recurrent tumor from patient #1 (FIGS. 3A-3B). In contrast, while H3K27ac increased slightly in the recurrent tumor from patient #2 and #3, the enrichment of H3K4me1 was not altered at their enhancer locus. Interestingly, we detected a significant increase in MGMT expression in the recurrent tumor from patient 1 compared to its matched primary tumor (FIG. 3C), whereas MGMT expression was not significantly altered in recurrent tumors from patients #2 and #3. Similarly, the fraction of MGMT expressing and proliferating tumor cells increased only in recurrent tumor from patient #1, while this fraction of cells was not observed in tumors from patient #2 and #3 (FIGS. 3D-3F, FIG. 10). Although the small sample size precludes robust conclusions, the data suggests activation of the novel enhancer may also drive the MGMT expression in some recurrent tumors with MGMT promoter methylation.

TABLE 4 Treatment information for patients who provided primary and recurrent tumor samples. Demographics Patient 1 Patient 2 Patient 3 Age 64 67 68 Sex Male Male Female Grade IV IV IV MGMT status methylated methylated methylated Treatment Subtotal Gross total Aggressive resection resection resection 60 Gy + TMZ 60 Gy + TMZ 60 Gy + TMZ adjuvant adjuvant adjuvant TMZ × 6 cyc TMZ × 6 cyc TMZ × 6 cyc Enrolled in N0877 trial 1^(st) disease free 4.7 mo 11.3 mo 15.6 mo interval* Salvage therapy TMZ × 6 cyc TMZ × 12 cyc TMZ × 6 cyc 2^(nd) disease free 0 mo 4 mo 0 mo interval** Treatment after 50 Gy + CCNU × 6 CCNU × 1 second TMZ mo Then cycle Then resection Then 3 35 Gy + CCNU & mo later Avastin Avastin × Avastin 8 mo Then 35 Gy Overall survival 29 months 55 months 43 months *Interval from the end of prior treatment to radiographic progression. **Interval from the end of prior treatment to resection. The patient demographics and treatment records were summarized in this table.

Example 5 Increased Acetylation at the Enhancer Increases MGMT Expression In Vitro

The level of H3K27ac at the enhancers is known to correlate with enhancer activity and gene expression. Therefore, we tested whether an increase in H3K27ac on nucleosomes surrounding the enhancer will affect MGMT expression by targeting the catalytic domain of histone acetyltransferase p300 to the enhancer locus (Hilton et al. 2015). Briefly, a Flag-tagged nuclease deactivated Cas9 (dCas9) protein was fused with p300 HAT domain and was targeted to the enhancer locus using five guide RNAs (gRNAs) in cells with low levels of MGMT expression (FIG. 4A). HEK293T cells were chosen first in this experiment due to their high transfection efficiency. Successful targeting of dCas9 or dCas9^(p300 Core) fusion proteins to the K-M enhancer locus was confirmed by Flag ChIP-qPCR (FIG. 4B). Moreover, we observed a marked increase of H3K27ac at the K-M enhancer locus when dCas9^(p300 Core) fusion protein, but not dCas9 alone, was expressed along with gRNAs (FIG. 4C). RT-PCR analysis indicated MGMT transcription also increased dramatically in HEK293T cells when targeting dCas9^(p300 Core) to the enhancer region by CRISPR/dCas9 (FIG. 4D). Although expression of the dCas9^(p300 Core) fusion protein alone without gRNAs slightly increased H3K27ac levels, this increase did not significantly alter MGMT expression, suggesting that a threshold level of H3K27ac is needed to alter the chromatin state of the enhancer and gene expression of MGMT. We also observed a slight, but significant increase in MGMT expression after we targeted dCas9^(p300 Core) to the enhancer locus in the 5199 line (FIGS. 4E-4G). Thus, the increased H3K27ac at nucleosomes surrounding the K-M enhancer can stimulate MGMT expression even in the presence of MGMT promoter methylation.

Example 6 Enhancer Depletion Reduces MGMT Expression and Increases TMZ Sensitivity

To directly determine whether the enhancer was essential for MGMT expression, we used the CRISPR/Cas9 system to delete a 3.3 kb region (chr10: 130,704,894-130,708,206) in the MGMT-expressing SKMG3 glioblastoma cell line (FIG. 5A) (Konermann et al. 2015; Ran et al. 2013). Four independent clones (three homozygous deletion clones and one wild-type control clone) were isolated. The genotypes of those four clones were identified by PCR and further confirmed by Sanger sequencing (FIGS. 11A-11B). Compared to parental SKMG3 cells and wild type control clone, MGMT transcription and protein expression level were markedly reduced in enhancer-deleted clones (FIGS. 5B-5C).

Next, we analyzed the TMZ sensitivity of SKMG3 clones using the clonogenic assay. Compared to parental SKMG3 lines and wild type control clone, the enhancer deleted clones were significantly more sensitive to TMZ treatment, with a dramatic log-fold decrease in IC₅₀ from over 100 μM to less than 10 μM (FIGS. 5D-5F). The sensitizing effect was not observed in the group pre-treated with the MGMT inhibitor O₆BG (FIGS. 5E-5F), indicating the change in TMZ sensitivity was mechanistically linked to MGMT activity. Since this TMZ concentration range is clinically achievable, these data suggest that efficient suppression of the enhancer activity could be a novel strategy to overcome the emergence of TMZ resistance.

To further narrow down the location of the enhancer activating the MGMT expression in SKMG3 cells, we designed an additional guide RNA (g3) targeted at the middle of the other two guide RNAs used for the 3.3 kb deletion and generated two different 1.5 kb deletions when combined with g1 or g2 guide RNAs, respectively (FIG. 12A). Depletion of the first 1.5 kb region (ch10: 130,704,894-130,706,549), named Del1 region, also resulted in a dramatic reduction in MGMT suppression in SKMG3 cells (FIG. 5G), while deletion of the Del2 region, the 1.8 kb region more closer to the MGMT promoter, did not cause a significant change in MGMT expression (FIG. 12B). This result further narrowed down the location of K-M enhancer to a 1.5 kb region that lies about 560 kb away from the MGMT promoter that is essential for MGMT expression. Similar to larger deletion clones, SKMG3 cells with the 1.5 kb deletion also showed increased TMZ sensitivity, but to lesser degree than deletion clones with the 3.3 kb region (FIG. 5H), suggesting that the second half of the 3.3 kb region also contributes to the regulation of MGMT expression. Finally, we deleted the 1.5 kb Del1 fragment from the 3080 line and obtained one clone (FIG. 12D). Deletion of this fragment in the 3080 cells also resulted in reduced MGMT expression (FIG. 5I) and increased TMZ sensitivity in vitro (FIG. 5J).

Example 7 Deletion of the Enhancer Leads to Impaired Proliferation and Reduced Expression of Ki67

K-M enhancer may not only regulate protein expression but also promotes proliferation. During clonogenic assay analysis, the untreated deletion clones formed smaller colonies suggestive of impaired proliferation (FIG. 11C). This observation was confirmed by a proliferation assay, showing a clear decrease in proliferation rate in two out of three 3.3 kb deletion and all 1.5 kb deletion SKMG3 clones (FIGS. 6A-6B, FIG. 12C). This suggests that the enhancer may also regulate expression of genes involved in proliferation in addition to MGMT. Because the enhancer resides between MGMT and MKI67, a gene encoding the nuclear protein Ki67 that serves as a proliferation marker for many tumors including GBM (FIG. 13A), we analyzed the expression of Ki67 in enhancer deleted clones. In SKMG3 enhancer deleted clones, the two 3.3 kb deletion clones which have reduced proliferation rates also showed a significant decrease in Ki67 expression (FIG. 6C). These same results were recapitulated in the 3080 Del1 enhancer deleted clone (FIGS. 6D-6E). Consistent with those findings, an increase in Ki67 was observed in the recurrent tumor from patient #1 with the activated enhancer (FIG. 13B). Collectively, our results indicate that the K-M enhancer, likely regulates both the expression of Ki67 and MGMT to control both cell proliferation and TMZ sensitivity.

Example 8 A New Plausible Strategy for the Design of Novel Biomarkers and Treatments Strategies for Cancers

Previous studies have associated MGMT hypermethylation with gene silencing (Watts et al. 1997). However, significant discordance between promoter hypermethylation and MGMT suppression are observed both in vitro and in vivo (Kitange et al. 2012; Wang et al. 1992). Here we identify a novel distal enhancer (K-M enhancer) regulating MGMT expression. Surprisingly, promoter hypermethylation does not suppress enhancer-mediated MGMT expression, suggesting the mechanism revealed in this study might contribute to the reported discordance between methylation and expression in at least a fraction of tumors. Deletion of the K-M enhancer reduces MGMT and Ki67 expression, decrease cell proliferation, and sensitizes cells to TMZ to a clinical relevant level, which suggests potential therapeutic benefits of targeting enhancer activity. Beyond MGMT, a set of 1141 novel putative enhancers were activated in the TMZ resistant 3080 line (Table 5, in which the genomic location of each putative enhancer in Group-1 cluster, shown in FIGS. 1A-1I, was listed. The H3K4me1 and H3K27ac change on each putative enhancer locus was also listed together with the calculation of significance. The mRNA ID for the RNA transcript of the gene closest to each putative enhancer was listed in the second column from the right. The fold change of expression for each transcript was listed in the first column for the right, calculated using the following formula: RNA-seq reads in 3080 line/RNA-seq reads in 5199 line.), which raises the possibility that multiple enhancers are altered in response to TMZ therapy and that more than one enhancer element may contribute to the emergence of drug resistance.

MGMT expression is mechanistically linked to TMZ resistance, and the discordance between promoter methylation and protein expression observed in a subset of patients limits the prognostic accuracy of methylation assessment. Despite the overt requirement for protein expression of MGMT to repair TMZ-induced damage, MGMT promoter methylation is a more accurate predictor of TMZ resistance as compared to either RNA or protein expression. However, the predicted favorable outcome was not observed in 25% of MGMT hypermethylated patients, who exhibited de novo resistance and was progressed within 9 month under TMZ therapy (Hegi et al. 2005). In this study, we demonstrate that activation of the K-M enhancer can drive high level MGMT expression despite promoter methylation. Moreover, a subset of MGMT hypermethylated GBM PDX models with K-M enhancer activation expresses basal MGMT protein in association with de novo TMZ resistance. These observations may help explain why approximately a quarter of newly diagnosed, MGMT hypermethylated GBM patients, progress within the first few months of TMZ therapy. Finally, the activation of the K-M enhancer during resistance emergence without corresponding changes to MGMT promoter methylation may partially explain the poor prognostic performance of methylation status in recurrent GBM. Although mechanisms of inherent and acquired TMZ resistance extend beyond MGMT regulation, we might anticipate that assessment of epigenetic states for both the promoter and enhancer may provide a more robust and accurate predictive biomarker for TMZ sensitivity.

Beyond enhanced prognostic accuracy, therapeutic suppression of K-M enhancer could be used to delay the emergence of TMZ resistance and/or sensitize resistant patients to TMZ. We showed that epigenetic activation of the K-M enhancer drives TMZ resistance, while enhancer deletion results in greater TMZ sensitivity. Similarly, blocking enhancer activation may prevent enhancer driven TMZ resistance. A similar concept has been tested in breast and lung cancer, where epigenetic inhibitors successfully prevented chemo-resistance emergence driven by epigenetic alterations of gene promoters (Gardner et al. 2017; Meisenberg et al. 2017). If successful, enhancer inhibition may prevent or delay the emergence of TMZ resistance and produce more durable responses for GBM patients, which may make a critical improvement in survival. Furthermore, deletion of the enhancer reduces proliferation and sensitizes cells to TMZ. This indicates that blocking K-M enhancer activity potentially not only enhances TMZ response, but also reduces aggressiveness of otherwise TMZ resistant tumors. In principle, enhancer activity can be blocked by several methods including blocking the recognition of acetylated histones and inhibition of histone acetyltransferase activity. For example, both Bromodomain inhibitors (BETi), which blocks H3K27ac recognition by Bromodomain containing proteins, and histone acetyltransferases inhibitors (HATi), which reduce enhancer activity by inhibiting H3K27 acetylation, can effectively inhibit enhancer-driven transcription activation with acceptable toxicity (Muller et al. 2011; Wadhwa et al. 2016; Milite et al. 2015). We have shown that a combination of p300/CBPi with TMZ resulted in reduced MGMT expression (FIGS. 15A and 15B) and increased TMZ sensitivity in vitro (FIGS. 16A and 16B). These results suggest that a combination of p300/CBPi or BETi with TMZ may delay the emergence of TMZ resistance in responsive tumors and potentially sensitize the drug resistant patients to TMZ.

In contrast to BET and HAT inhibitors, histone deacetylase inhibitors (HDACi), which globally increase histone acetylation including H3K27ac, may promote the emergence of TMZ resistance by activating the K-M enhancer. Supporting this ides, we observed that combination treatment of SAHA, an FDA-approved HDACi, with TMZ specifically promotes elevation of MGMT expression as a mechanism of TMZ resistance (Kitange et al. 2012). Our enhancer activation model suggests that acetylation of both the MGMT promoter and the K-M enhancer could be a major cause of this effect. Thus, any future designs of treatment strategies that combine TMZ and histone deacetylase inhibitors should be approached with appropriate caution.

Beyond mediating MGMT expression and TMZ sensitivity, enhancer deletion was associated with significantly reduced proliferation in five out of six enhancer deleted clones, suggesting that K-M enhancer inactivation may also reduce tumor growth. Interestingly, unlike the observed increase in MGMT expression, Ki67 expression in 5199 was similar to 3080 line, indicating differential influence of the K-M enhancer on the two most proximal genes, MGMT and MKI67. One possible explanation for this would be if the enhancer-promoter interactions for MGMT and MKI67 are mediated by different transcription factors. Alternatively, it is possible that some PDX lines including 3080 have sequence changes at this K-M enhancer region. This change either creates a binding site for transcription activators or destroys a binding site for transcription repressors. Future studies are needed to catalog the transcription factors that bind to the K-M enhancer. Taken together, the K-M enhancer inhibition may not only reduce MGMT expression but also reduce tumor aggressiveness.

In summary, our study reveals a previously undocumented enhancer that, when activated, promotes MGMT expression and TMZ resistance in different cell lines, even in the presence of promoter methylation. This enhancer also regulates the expression of Ki67 to influence tumor aggressiveness. Loss of this enhancer is associated with increased TMZ sensitivity and reduced proliferation. These findings suggest that inhibition of enhancer activity is a new plausible strategy for the design of novel biomarkers and treatments strategies for cancers.

TABLE 5 The original list of Group-1 putative enhancers. chromo- H3K4me1 H3K27ac RNAseq some star end strand (3080/5199) p-value (3080/5199) p-value adjacent gene (3080/5199) chr10 1.31E+08 1.31E+08 + 2.63 0.27294 3.35 7.28E−15 NM_002412 15.63 chr10 1.31E+08 1.31E+08 + −0.04 0.956481 4.52 5.98E−07 NM_002412 15.63 chr17 15157604 15157732 + 0.57 0.230388 2.38 4.45E−08 NR_039884 7.57 chr19 13974673 13974801 + 0.13 0.464479 2.41 3.22E−05 NR_029613 7.49 chr1 84012538 84012744 + 0.23 0.02581 1.98 7.12E−06 NR_049805 6.64 chr11 75051486 75052116 + 1.04 0.06437 1.37 2.17E−08 NR_029891 6.13 chr13 80380579 80380870 + −0.68 0.029802 1.49 2.76E−05 NR_046685 5.91 chr8 53341449 53341663 + 2.26 0.065139 2.64 2.45E−06 NM_014682 5.64 chrY  6331521  6331671 + 0.96 0.003193 4.4 2.04E−06 NR_003591 4.91 chr10 49916455 49916676 + 2.28 0.010657 2.34 1.37E−05 NM_020945 4.32 chr17 34092496 34092624 + 0.92 0.152933 2.15 9.64E−05 NM_145272 4.32 chr4 54501374 54501502 + 1.26 0.156387 2.88 3.48E−05 NR_046827 4.32 chr7 69852399 69852616 + 0.81 0.93262 2.11 3.97E−05 NM_022479 4.32 chr1 18014635 18014807 + 2.04 0.099329 3.05 2.00E−07 MM_030812 3.32 chr1 2.34E+08 2.34E+08 + 1.38 0.002207 1.7 8.33E−11 NM_032435 3.22 chr1 2.34E+08 2.34E+08 + 2.16 0.016043 2.17 1.66E−06 NM_032435 3.32 chr10 98878556 98876785 + −1.54 0.103108 3.38 1.29E−07 NR_038330 3.32 chr10 1.07E+08 1.07E−08 + 0.52 0.044377 1.36 4.82E−15 NM_014978 3.32 chr17  1157260  1157466 + −0.96 0.104912 2.34 1.37E−05 NM_001164405 3.32 chr18 29161447 29161805 + −0.03 0.460491 1.83 1.43E−07 NM_000371 3.32 chr18 40238107 40238433 + 0.63 0.261697 2.39 1.03E−11 NM_002930 3.32 chr18 53958769 53958980 + 0.23 0.391248 1.62 3.67E−05 NR_040026 3.22 chr2  1.5E+08  1.55+08 + 0.46 0.463277 1.77 4.13E−05 NM_177964 3.32 chr20 45192532 45192824 + −0.93 3.038183 1.48 4.35E−07 NM_080721 3.32 chr20 46756813 46757250 + −0.3 0.29184 1.33 2.91E−06 NR_228958 3.32 chr20 46831807 46831971 + 0.46 0.463277 3.46 4.08E−08 NR_026658 3.32 chr3 38786368 38786977 + 3.26 2.23E−05 2 2.30E−11 NM_006514 3.32 chr4 94337656 94338038 + −1.13 0.108085 1.71 2.84E−06 NM_005172 3.32 chr5 56752078 56752404 + −0.28 0.353275 2.2 2.71E−07 NM_001017992 3.32 chr5 56790567 56791307 + 1.04 0.013395 1.38 2.29E−08 NM_001017992 3.32 chr5 56797905 50798110 + 1.26 0.156387 2.01 4.17E−05 NM_001017992 3.32 chr9 1.04E+08 1.04E+08 + 1.78 0.052385 1.54 1.27E−05 NM_147180 3.32 chr8 1.44E+08 1.44E+98 + −1.13 0.065395 2.72 8.55E−07 NM_001702 3.17 chr19  8131466  8131777 + −0.65 0.045532 1.33 8.07E−06 NM_005624 3 chr17   45209   45345 + −0.04 0.956481 2.81 5.99E−05 NM_003585 2.74 chr17 81059143 81059279 + 1.46 0.222672 4.2 1.27E−05 NM_001004431 2.32 chr7 43139789 43140395 + 0.98 0.049001 2.08 2.70E−16 NM_015052 2.32 chr7 43147941 43148069 + −0.77 0.876277 2.13 6.02E−07 NM_015052 2.32 chr7 43151077 43151744 + 1.3 0.0345 2.13 1.38E−12 NM_015052 2.32 chr10 15335805 15337012 + 2.7 1.43E−10 2.82 3.92E−27 NM_001010924 2.2 chr10 15387046 15387750 + 2.39 7.41E−08 1.61 3.41E−09 NM_001010924 2.2 chr10 15394883 15395011 + −0.54 0.361994 2.26 1.64E−07 NM_001010924 2.2 chr10 15409910 15410105 + −1.13 0.188905 2.73 4.67E−10 NM_001010924 2.2 chr5  1.7E+08  1.7E+08 + −0.77 0.876277 4.34 3.77E−06 NM_001034837 2.2 chr5  1.7E+08  1.7E+08 + 0.73 0.02296 2.44 3.81E−13 NM_001034837 2.2 chr5  1.7E+08  1.7E+08 + 0.52 0.012348 1.31 2.22E−16 NM_001034837 2.2 chr5  1.7E+08  1.7E+08 + 1.46 0.010305 1.7 9.76E−05 NM_001034837 2.2 chr5  1.7E+08  1.7E+08 + 0.46 0.463277 2.31 8.78E−05 NM_001034837 2.2 chr5  1.7E+08  1.7E+08 + −0.22 0.311924 1.38 1.96E−10 NM_001034837 2.2 chr5  1.7E+08  1.7E+08 + 1.99 0.000726 2.24 1.36E−10 NM_001034837 2.2 chr5  1.7E+08  1.7E+08 + 1.59 0.000322 1.51 1.54E−10 NM_001034837 2.2 chr5  1.7E+08  1.7E+08 + 2.63 0.027294 1.6 9.18E−06 NM_001034837 2.2 chr3 1.35E+08 1.35E+08 + −0.54 0.329466 1.73 1.88E−05 NM_004441 1.91 chr4 1.41E+08 1.41E+06 + 2.46 0.042327 2.46 3.03E−06 NM_018717 1.91 chr15 69085288 69085426 + −0.13 0.374716 2.29 2.24E−05 NR_036197 1.9 chr15 69094025 69094269 + 0.04 0.492338 1.84 4.25E−05 NR_036197 1.9 chrX 1.33E+08 1.33E+08 + 0.46 0.463277 2.65 2.30E−07 NM_001164617 1.86 chrX 1.33E+08 1.33E+08 + −0.19 0.011157 3.2 7.38E−05 NM_001164617 1.86 chrX 1.33E+08 1.33E+08 + 0.78 0.301098 1.63 2.81E−07 NM_001164617 1.86 chrX 1.33E+08 1.33E+08 + 0.23 0.02581 2.11 397E−05 NM_001164617 1.86 chr12 15627091 15627820 + 2.26 0.000362 1.34 7.42E−07 NM_030671 1.83 chr4 1.58E+08 1.58E+08 + 4.73 5.59E−23 2.28 3.38E−16 NM_001083620 1.78 chr4 1.58E+08 1.58E+08 + 2.04 0.099329 3.68 7.23E−07 NM_001083620 1.78 chr4 1.58E+08 1.58E+08 + 0.78 0.077498 1.4 2.80E−07 NM_001083620 1.78 chr12 15508238 15508399 + 0.23 0.02581 3.29 4.03E−07 NM_030667 1.7 chr12 15504789 15510038 + 1.54 0.022876 2.71 7.11E−11 NM_030667 1.7 chr12 15570562 15571563 + 1.84 2.03E−05 1.65 2.72E−13 NM_030667 1.7 chr12 15574316 15574515 + 1.16 0.08465 3.92 3.74E−08 NM_030667 1.7 chr12 15575037 15575342 + 1.23 0.001123 1.88 4.69E−06 NM_030667 1.7 chr16 81674249 81674377 + −2.13 0.027712 4.2 1.27E−05 NR_045112 1.58 chr8 1.05E+08 1.05E+08 + 1.26 0.156387 1.71 7.60E−07 NM_001385 1.58 chr6 1.23E+08 1.23E+08 + 0.84 0.148331 2.36 6.32E−10 NM_001010852 1.52 chr6 1.23E+08 1.23E+08 + −0.77 0.876277 2.19 3.14E−07 NM_001010852 1.52 chr6 1.23E+08 1.23E+08 + 0.8 0.05977 1.79 3.37E−07 NM_001010852 1.52 chr16 55840982 55841158 + 1.54 0.022876 2.05 2.62E−05 NM_001025194 1.5 chr5 74173734 74173922 + −0.25 0.332095 2.06 6.83E−06 NM_015586 1.47 chrX 1.33E+08 1.33E+08 + 1.78 0.003194 1.98 6.77E−15 NM_001448 1.46 chrX 1.33E+08 1.33E+08 + 0.19 0.465306 1.82 4.67E−06 NM_001448 1.46 chrX 1.33E+08 1.33E+08 + 2.04 0.099329 1.9 5.25E−07 NM_001448 1.46 chr8  4621157  4621293 + 1.34 0.000666 2.26 1.64E−07 NM_033225 1.42 chr21 40359192 40360744 + 0.68 0.026258 1.68 1.02E−17 NM_005239 1.37 chr21 40362390 40362524 + −0.22 0.38435 2.88 3.48E−05 NM_005239 1.37 chr1 17873576 17873755 + 0.35 0.325791 1.95 2.71E−05 NM_018125 1.36 chr20 36023210 36023597 + −0.37 0.217565 1.67 2.51E−09 NM_198291 1.36 chr4 40850203 40851064 + 2.32 1.41E−09 1.36 1.08E−09 NM_001168051 1.36 chr4 40851715 40851899 + 3.16 0.004461 2.24 9.42E−05 NM_001166051 1.36 chr4 40856855 40857235 + 1.7 0.01132 2.05 3.14E−07 NM_001186051 1.36 chr1 17892110 17892281 + 0.68 0.280156 1.68 6.15E−05 NM_001011722 1.35 chr1 17895787 17896416 + 1.25 0.000103 1.92 3.42E−13 NM_001011722 1.35 chr1 17900076 17900242 + 1.64 0.000139 3.01 3.58E−07 NM_001011722 1.35 chr1 17913886 17914567 + 1.87 5.91E−09 1.31 6.83E−09 NM_001011722 1.35 chr17 71477113 71477829 + 0.56 0.128974 2.43 2.81E−15 NM_001144952 1.33 chr17 71538702 71538899 + 1.38 0.025068 4.27 1.65E−10 NM_001144952 1.33 chr17 71546866 71547022 + 1.63 0.07633 3.29 4.03E−07 NM_001144952 1.33 chr17 71637475 71637752 + −0.54 0.3033 2.17 4.36E−07 NM_001144952 1.33 chr11 34294794 34295009 + 1.26 0.061907 2.2 3.96E−08 NM_145804 1.28 chr11 34321286 34321437 + 0.46 0.404925 2.29 2.24E−05 NM_145804 1.26 chr4 37931201 37931609 + −0.19 0.011157 1.49 2.76E−05 NM_006607 1.26 chr1 2.31E+08 2.31E+08 + 0.88 0.126482 1.87 3.59E−18 NM_001258311 1.22 chr1 2.31E+08 2.31E+08 + 0.04 0.504309 1.76 7.78E−10 NM_001258311 1.22 chr1 2.31E+08 2.31E+08 + 0.52 0.088482 1.42 8.20E−13 NM_001258311 1.22 chr20 50386254 50386386 + 0.87 0.210905 3.05 6.68E−06 NM_006045 1.21 chr3 25513336 25515881 + 0.81 0.000325 1.71 2.48E−31 NM_000965 1.18 chr16 84873786 84873915 + 0.46 0.463277 2.52 7.56E−05 NM_031476 1.17 chr3 63398978 63399527 + 0.22 0.369908 1.59 1.18E−08 NM_144642 1.12 chrX 1.33E+08 1.33E+08 + 0.81 0.005284 2.86 6.62E−11 NM_001101357 1.12 chrX 1.33E+08 1.33E+08 + 0.68 0.280156 2.25 3.66E−05 NM_001101357 1.12 chr6 71744515 71744826 + −0.28 0.353275 1.39 4.86E−05 NM_080742 1.11 chr6 71790599 71790937 + 0.33 0.277101 1.49 1.73E−05 NM_080742 1.11 chr8 1.01E+08 1.01E+08 + −0.18 0.381896 1.78 1.92E−06 NM_003114 1.11 chr12 94441501 94441629 + −0.54 0.361994 4.27 6.94E−06 NM_005761 1.08 chr22 26663920 26664258 + −0.42 0.23405 1.46 1.59E−05 NM_001184774 1.06 chr6 94118302 94118451 + −0.06 0.44608 3.2 1.25E−06 NM_004440 1.03 chr17  3864193  3664479 + −1.13 0.188905 1.85 1.26E−08 NM_005173 1 chr18 54540803 54541253 + −0.31 0.249034 1.67 9.22E−09 NR_048536 1 chr8 70378884 70279103 + −0.54 0.361994 3.08 3.76E−09 NM_001128204 1 chr8 70398889 70399134 + 0.81 0.93262 3.91 1.99E−11 NM_001128205 1 chr8 70399756 70399884 + 0.23 0.02581 2.49 1.21E−08 NM_001128205 1 chr2 1.05E+08 1.05E+08 + 0.46 0.308555 1.31 3.20E−05 NR_015399 0.97 chr2 1.08E+08 1.08E+08 + 0.78 0.391098 2.52 7.56E−05 NM_001142352 0.94 chr8 1.22E+08 1.22E+08 + 1.38 0.025068 2.03 1.32E−07 NM_021021 0.93 chr8 1.22E+08 1.22E+08 + 0.75 0.100892 1.79 7.19E−06 NM_021021 0.93 chr8 1.22E+08 1.22E+08 + −0.41 0.248943 2.12 1.03E−05 NM_021021 0.93 chr3 69869675 69870370 + 1.19 0.01248 2.28 3.38E−16 NM_198177 0.91 chr3 69870955 69871136 + 0.92 0.152933 4.05 6.20E−09 NM_198177 0.91 chr16  8635808  8636226 + 0.92 0.013438 1.31 3.20E−05 NM_001146336 0.89 chr21 33368894 33369446 + 1.63 0.00023 1.47 4.57E−09 NR_038033 0.87 chr21 33515567 33515782 + −2.28 0.004579 3.01 3.58E−07 NR_038033 0.87 chr1 1.12E+08 1.12E+08 + −0.83 0.033279 1.48 4.35E−07 NM_172198 0.86 chr9 1.03E+08 1.03E+08 + 1.78 0.149771 2.93 2.01E−05 NM_001018116 0.86 chr9 1.03E+08 1.03E+08 + −0.63 0.10124 2.06 8.33E−08 NM_001018116 0.86 chr2 84179635 84179923 + 1.04 0.010434 1.71 2.64E−06 NR_003663 0.85 chr5 13990775 13991016 + −0.08 0.429403 2.1 4.28E−06 NM_001369 0.85 chr9 1.13E+08 1.13E+08 + −1.09 0.016514 2.38 8.30E−06 NM_001012993 0.85 chr12 14075473 14075863 + 0.92 0.063595 1.35 2.50E−05 NM_000834 0.84 chr2 40447265 49447617 + 0.11 0.462988 2.38 5.51E−08 NM_001112800 0.83 chr2 40599364 40599574 + 0.87 0.210905 3.61 4.01E−09 NM_001112800 0.83 chr2 40662185 40662518 + 0.46 0.365076 2.82 9.15E−10 NM_001112800 0.83 chr5 64437302 64437564 + 0.46 0.463277 1.38 7.74E−05 NM_197941 0.81 chr2  5532734  5532957 + 3.21 3.53E−05 1.64 0.000358 NM_003108 0.8 chr4 81120300 81120428 + 0.45 0.015307 2.63 2.67E−05 NM_001099403 0.8 chr10 1.02E+08 1.02E+08 + −0.36 0.926622 1.68 6.15E−05 NM_003988 0.79 chr10 1.24E+08 1.24E+08 + 0.46 0.308555 1.84 4.25E−05 NM_002775 0.79 chr21 33263348 33263653 + 1.13 0.063706 1.73 1.68E−05 NM_014586 0.78 chr8 1.26E+08 1.26E+08 + 0.37 0.283356 2.2 1.53E−05 NM_014751 0.78 chr20 46391324 46392013 + 0.04 0.477655 1.39 8.78E−11 NM_018837 0.75 chr6  1.3E+08  1.3E+08 + 1.26 0.156387 3.52 4.18E−06 NM_033515 0.75 chr22 27459620 27460046 + 1.54 0.022876 2.1 3.04E−11 NR_003491 0.74 chr22 27469882 27470264 + 2.36 0.007037 1.6 4.58E−08 NR_003491 0.74 chr22 27504268 27505353 + 0.56 0.077732 1.61 2.35E−13 NR_003491 0.74 chr22 27507214 27507633 + 2.96 7.37E−06 1.51 4.68E−06 NR_003491 0.74 chr22 27511555 27512120 + 1.11 0.036985 1.9 1.63E−08 NR_003491 0.74 chr4 55540038 55540166 + −0.96 0.18457 3.2 7.36E−05 NM_000222 0.74 chr11 89802126 69803147 + 1.48 3.76E−05 1.53 4.00E−14 NM_018043 0.73 chr20 46423943 46424275 + 0.13 0.429293 1.4 1.21E−05 NM_198596 0.73 chr20 46505367 46505541 + 0.46 0.404925 1.7 2.56E−05 NM_198596 0.73 chr17 19170339 19170482 + 0.46 0.334119 2.78 5.42E−06 NR_046824 0.68 chr17 19182076 19182216 + 0.46 0.221407 1.96 6.59E−05 NR_046824 0.68 chr17 19182743 19183382 + 2.2 1.93E−10 1.42 1.23E−07 NR_046824 0.68 chr18  9813187  9813315 + −0.42 0.23405 2.01 4.17E−05 NM_032243 0.68 chr2 1.86E+08 1.86E+08 + 1.81 0.834416 2.13 6.02E−07 NM_194250 0.67 chr1 2.43E+08 2.43E+08 + 0.4 0.480721 1.38 1.96E−10 NM_001195811 0.66 chr1 2.43E+08 2.43E+08 + 3.82 6.26E−05 1.61 2.31E−05 NM_001195811 0.68 chr14 47861658 47861890 + −0.25 0.332095 1.48 1.09E−05 NM_182830 0.66 chr14 47936164 47936721 + 0.54 0.111688 1.48 6.99E−08 NM_182830 0.66 chr14 47970662 47977456 + 0.49 0.115226 1.42 3.11E−10 NM_182830 0.68 chr9 1.09E+08 1.09E+08 + −0.96 0.18457 3.05 6.68E−05 NM_021224 0.65 chr5 1.47E+08 1.47E+08 + 1.7 5.10E−08 1.33 1.19E−19 NR_073101 0.63 chr5 1.47E+06 1.47E+08 + 1.96 0.009351 1.88 3.41E−07 NR_073101 0.63 chr5 1.47E+08 1.47E+08 + 2.98 1.68E−07 1.85 6.47E−14 NR_073101 0.63 chr5 1.47E+08 1.47E+08 + 1.14 4.75E−06 1.55 3.36E−25 NR_073101 0.63 chr5 1.47E+08 1.47E+08 + 0.29 0.337406 1.38 6.92E−06 NR_073101 0.63 chr5 1.47E+08 1.47E+08 + 0.19 0.465306 2.34 2.39E−08 NR_073101 0.63 chr5 1.47E+08 1.47E+08 + 0.81 0.005384 2.86 6.02E−11 NR_073101 0.63 chr4 76955564 76956038 + 1.08 0.013198 1.6 4.58E−08 NM_005409 0.59 chr20 50582889 50583023 + 1.63 0.07633 1.91 1.75E−05 NM_020436 0.58 chr4 41543912 41544081 + 1.84 0.000139 2.15 9.64E−05 NM_001112719 0.58 chr5  7759550  7759941 + 1.65 0.028759 1.88 1.43E−07 NM_001089584 0.58 chr5 1.45E+08 1.45E+08 + 1.13 0.012899 2.3 1.03E−08 NM_182960 0.58 chr5 1.45E+08 1.45E+08 + 0.80 0.000372 1.72 7.66E−13 NM_182960 0.58 chr7 19955874 19956060 + 0.23 0.02581 2.05 2.62E−05 NM_152774 0.58 chr7 1.01E+08 1.01E+08 + −0.19 0.01157 3.1 3.83E−06 NM_003378 0.58 chr9 1.37E+08 1.37E+08 + 1.73 3.53E−05 1.68 1.62E−05 NM_007101 0.58 chr2 2.22E+08 2.22E+08 + 1.92 0.035602 2.07 6.37E−05 NM_004438 0.57 chr2 2.22E+08 2.22E+08 + 1.41 0.004736 1.59 5.79E−06 NM_004438 0.57 chr12 1.33E+08 1.33E+08 + −1.28 0.039667 2.39 2.01E−05 NM_001142641 0.56 chr10 15279576 15279729 + −0.54 0.361994 3.05 6.78E−06 NM_004808 0.55 chr11  7593182  7593676 + 2.23 4.07E−06 2.05 1.60E−12 NM_001256568 0.55 chr11  7600776  7601522 + 1.32 0.000664 2.1 3.04E−11 NM_001256568 0.55 chr11  7602134  7602568 + 3.26 2.23E−05 3 2.07E−11 NM_001256568 0.55 chr11  7603953  7604122 + 1.64 0.000139 2.41 3.22E−05 NM_001256568 0.55 chr11  7608273  7609591 + 2.77 4.01E−22 2.04 1.14E−31 NM_001256568 0.55 chr11  7651020  7051846 + 3.90 9.47E−13 1.9 2.45E−16 NM_001256569 0.55 chr11  7656210  7656676 + 1.73 0.001876 2.27 4.50E−14 NM_001256569 0.55 chr11 41399978 41400649 + 0.84 0.035899 1.31 5.42E−07 NM_001258419 0.55 chr4 1.21E+08 1.21E+08 + 0.94 0.113163 1.73 1.87E−06 NM_033430 0.55 chr4 1.21E+08 1.21E+08 + −0.22 0.38435 3.2 1.25E−06 NM_001083 0.55 chr16 77849568 77849832 + 1 0.022386 2.09 4.71E−07 NM_020927 0.54 chr16 77884557 77884685 + 1.04 0.218994 3.4 1.33E−05 NM_020927 0.54 chr22 27643727 27643911 + 0.46 0.463277 2.72 8.55E−07 NM_002430 0.54 chr22 27812662 27812979 + 0.04 0.529972 1.95 2.15E−07 NM_002430 0.54 chr22 28144856 28146329 + 0.16 0.427697 1.5 4.71E−07 NM_002430 0.54 chr8 97539032 97539160 + 1.04 0.156144 2.83 3.17E−06 NM_002998 0.54 chr18 43989632 43989832 + 3.36 0.00176 2.32 3.54E−07 NM_152470 0.52 chr7 15819961 15820159 + 0.66 0.121103 1.7 6.77E−06 NM_005924 0.52 chr8 1.21E+08 1.21E+08 + 1.22 0.007309 2.49 2.88E−07 NM_014078 0.52 chr8 1.22E+08 1.22E+08 + 0.52 0.08078 2.12 7.79E−08 NM_014078 0.52 chr18 13229002 13229379 + 0.16 0.381985 1.31 2.62E−06 NM_181482 0.51 chr2 74518776 74519077 + 0.92 0.152933 1.9 1.26E−06 NM_021196 0.51 chr5 15260987 15261346 + −0.54 0.245002 1.72 4.46E−06 NM_012304 0.51 chr8 70699186 70699321 + 1.19 0.113922 3.63 1.30E−06 NM_001146009 0.51 chr9 1.36E+08 1.36E+08 + −0.22 0.38435 1.43 9.39E−06 NM_001042368 0.51 chr9 1.36E+08 1.36E+08 + 1.28 0.156387 2.05 2.62E−05 NM_001042368 0.51 chr13 25709818 25710693 + 0.38 0.265527 1.93 1.05E−11 NM_199138 0.5 chr7 1.05E+08 1.05E+08 + 0.19 0.396995 2.07 6.37E−05 NM_152749 0.5 chr7 1.06E+08 1.06E+08 + −0.06 0.44608 2.51 1.16E−05 NM_152749 0.5 chr8 22927847 22928059 + 1.63 0.07633 2.49 2.88E−07 NM_003842 0.5 chrX  1.3E+08  1.3E+08 + 0.81 0.079776 1.55 5.14E−05 NM_182314 0.5 chrX  1.3E+08  1.3E+08 + 1.7 0.01132 3.19 1.89E−13 NM_182314 0.5 chrX  1.3E+08  1.3E+08 + 2.92 0.011142 2.63 2.67E−05 NM_182314 0.5 chrX  1.3E+08  1.3E+08 + 3.54 0.000686 2.86 6.62E−11 NM_182314 0.5 chrX  1.3E+08  1.3E+08 + 1.78 0.003194 2.11 3.97E−05 NM_182314 0.5 chr17 20825376 20825725 + 0.68 0.126482 1.44 5.78E−06 NM_001004306 0.49 chr3 1.11E+08 1.11E+08 + −0.36 0.926622 2.68 1.45E−06 NR_045114 0.49 chr3 1.38E+08 1.38E+08 + 1.28 1.97E−09 1.61 1.88E−20 NM_001252090 0.49 chr5 1.54E+08 1.54E+08 + 1.25 0.000215 1.71 4.57E−25 NM_198321 0.49 chr7 87142891 87143294 + 1.63 0.07633 1.4 1.21E−05 NM_018849 0.49 chr8 85165622 85165750 + 2.26 0.065139 3.2 7.38E−05 NM_001100391 0.48 chr9 19481585 19482028 + 1.13 0.063706 1.6 1.46E−05 NM_001010887 0.48 chr13 78762313 78762466 + 1.04 0.325105 2.41 3.22E−05 NR_047001 0.47 chr2  1613290  1613612 + 0.19 0.424653 1.39 2.76E−06 NM_012293 0.47 chr8 1.21E+08 1.21E+08 + −0.81 0.170714 2.52 2.77E−08 NM_021110 0.47 chr8 1.21E+08 1.21E+08 + −0.04 0.956481 2.03 1.19E−06 NM_021110 0.47 chr4 73253915 73254058 + −0.11 0.394245 1.88 6.65E−05 NM_014243 0.46 chr4 73290340 73290510 + −1.02 0.010478 1.7 9.76E−05 NM_014243 0.46 chr7 55135496 55135624 + 1.16 0.000651 1.51 1.48E−09 NM_201284 0.46 chr16 11766248 11766572 + 0 0.521069 1.58 3.45E−05 NM_003498 0.45 chr19 46907361 46907664 + 0.67 0.062848 1.76 1.23E−06 NM_032040 0.45 chr19 46929003 46929166 + 1.46 0.032155 2.8 2.95E−07 NM_032040 0.45 chr19 46937902 46938046 + 1.46 0.109973 2.52 7.56E−05 NM_032040 0.45 chr12 28947573 28947924 + −0.06 0.437036 1.68 1.62E−05 NM_001271783 0.44 chr12 29395381 29395540 + 0.04 0.504309 3.09 1.18E−07 NM_018099 0.44 chr3 1.27E+08 1.27E+08 + 0.78 0.201565 2.41 3.22E−05 NM_032242 0.44 chr3 1.27E+08 1.27E+08 + −0.54 0.329468 4.52 5.98E−07 NM_032242 0.44 chr4 77817592 77817834 + −0.54 0.19189 2.98 1.96E−08 NM_001029870 0.44 chr5  9482727  9484410 + 1.12 1.00E−05 1.91 4.64E−29 NM_003966 0.44 chr8 1.46E+08 1.46E+08 + 0.46 0.334119 2.15 9.64E−05 NM_213605 0.44 chr11 1.17E+08 1.17E+08 + −0.23 0.201545 1.31 2.62E−08 NM_020693 0.43 chr10 60410762 60411324 + 0.9 0.083608 2.12 7.11E−11 NR_027506 0.42 chr10 60580636 60580764 + −0.77 0.876277 3.2 7.38E−05 NR_027508 0.42 chr18 22846936 22847453 + 0.94 0.005082 1.68 3.93E−09 NM_015461 0.42 chr18 22878853 22879021 + 0.46 0.286689 1.39 4.86E−05 NM_015461 0.42 chr19 35547644 35547696 + 0.46 0.463277 2.2 3.96E−05 NM_182983 0.42 chr21 34616788 34618980 + 0.29 0.282308 2.25 3.66E−05 NM_207585 0.42 chr3 1.34E+08 1.34E+08 + 0.75 0.100892 2.39 1.31E−06 NM_016201 0.42 chr9 1.19E+08 1.19E+08 + 2.04 0.008345 1.86 1.64E−08 NM_198186 0.41 chr18 56743151 56743384 + −0.54 0.162324 2.56 1.60E−09 NR_024021 0.4 chr9 46115227 46115417 + 0.48 0.150594 2.11 3.97E−05 NR_103712 0.4 chr3 1.73E+08 1.73E+08 + −2.54 0.044242 2.41 3.22E−05 NM_014932 0.39 chr3 1.73E+08 1.73E+08 + 0.17 0.379053 1.5 2.98E−07 NM_014932 0.39 chr16 27793865 27794030 + −0.17 0.344028 2.2 5.95E−05 NM_144675 0.38 chr3 1.53E+08 1.53E+08 + −0.68 0.134838 1.48 4.35E−07 NM_002563 0.38 chr3 1.53E+08 1.53E+08 + 0.04 0.529972 2.25 3.66E−05 NM_002563 0.38 chr7 55320652 55320780 + −0.81 0.006997 1.6 2.91E−08 NR_047551 0.38 chr9 1.246+08 1.24E+08 + 1.63 0.07633 2.52 7.56E−05 NM_138709 0.38 chr1 2.08E+08 2.08E+08 + 2.26 0.010657 1.67 1.97E−07 NM_025179 0.37 chr2 2.02E+08 2.02E+08 + 1.78 0.01992 2.36 5.33E−05 NM_001142356 0.37 chr3 29502406 29502534 + −0.06 0.44608 2.43 2.32E−08 NM_001177712 0.37 chr11 85251497 85251925 + 2.16 0.000802 1.5 7.44E−07 NM_001142699 0.36 chr16  726153  726367 + 1.78 0.01992 2.41 3.22E−05 NM_003961 0.36 chr18 25597547 25597712 + 1.04 0.085353 2.54 1.09E−03 NM_001792 0.36 chr18 25613369 25813506 + 0.78 0.201565 1.95 2.71E−05 NM_001792 0.36 chr18 25729930 25730338 + 0.22 0.252701 1.39 4.13E−08 NM_001792 0.36 chr18 26470533 26471078 + 2.57 0.00024 1.72 9.93E−09 NM_001792 0.36 chr2 12883536 12883741 + −0.21 0.309874 1.63 5.84E−05 NR_036072 0.36 chr22 39644794 39644930 + −1.54 0.103108 3.05 6.68E−06 NM _002608 0.36 chr22 39650793 39651509 + 1.29 5.83E−05 1.36 5.78E−12 NM_002608 0.36 chr3 1.12E+08 1.12E+08 + −0.06 0.44608 2.34 1.37E−05 NM_199512 0.36 chr8 1.45E+08 1.45E+08 + 3.54 0.000686 2.68 1.57E−05 NM_015117 0.36 chr9 80512297 80512736 + −0.28 0.310976 1.3 1.03E−05 NM_00272 0.36 chr9 1.27E+08 1.27E+08 + 1.88 0.000267 1.48 2.73E−06 NR_038975 0.36 chr9 1.34E+08 1.34E+08 + 0.58 0.142237 1.4 2.62E−08 NM_033387 0.36 chr2 1.66E+08 1.66E+08 + −0.54 0.261994 2.78 2.43E−10 NM_001172173 0.35 chr7  196138  156498 + 0.78 0.077498 1.98 6.53E−09 NM_020223 0.35 chr11 33497653 33498339 + 0.39 0.249424 1.88 8.33E−13 NM_012194 0.34 chr11 33502186 33502435 + 0.78 0.301098 1.92 4.25E−05 NM_012194 0.34 chr12 64291605 64292134 + 0.46 0.136027 1.38 4.02E−07 NM_020762 0.34 chr12 64303495 64303644 + −0.87 0.178044 2.25 3.66E−05 NM_020762 0.34 chr12 64311063 64312478 + 0.74 0.006402 1.34 1.59E−12 NM_020762 0.34 chr12 64338601 64338763 + 1.23 0.001123 3.46 4.08E−08 NM_020762 0.34 chr2 70985550 70985924 + −0.2 0.353044 1.9 2.20E−07 NM_001185054 0.34 chr10 1.03E+08 1.03E+08 + 1.19 0.001665 1.43 1.38E−10 NM_018121 0.33 chr12 1.23E+08 1.23E+08 + 0.35 0.325791 2.51 1.16E−05 NM_003959 0.33 chr13 44336319 44336853 + 2.23 2.13E−06 3.01 2.28E−14 NM_001242863 0.33 chr13 44337500 44337834 + 0.46 0.286689 1.93 6.73E−09 NM_001242863 0.33 chr13 44338512 44339608 + 1.81 6.81E−05 2.35 1.71E−21 NM_001242863 0.33 chr13 44341224 44341935 + 2.7 2.75E−08 1.83 1.81E−14 NM_001242863 0.33 chr13 44352470 44352671 + 3.04 0.007085 3.58 7.18E−09 NM_001242863 0.33 chr2 65641494 65641893 + 0.94 0.037428 1.58 3.95E−07 NM_181784 0.33 chr8 1.19E+08 1.19E+08 + −0.67 0.128489 1.97 2.96E−06 NM_000127 0.33 chr10 95854338 95854466 + −0.54 0.261994 3.4 1.33E−05 NM_001165979 0.32 chr10   1E+08   1E+08 + 2.78 0.017487 1.68 6.15E−05 NM_032211 0.32 chr14   1E+08   1E+08 + 2.26 0.065139 2.52 7.56E−05 NM_001144995 0.32 chr15 35475360 35475517 + −0.96 0.18457 4.96 2.25E−09 NR_003144 0.32 chr22 38662786 38663074 + 0.68 0.090078 1.41 8.54E−05 NM_001195071 0.32 chr6 17698540 17698774 + −0.42 0.23405 1.84 4.25E−05 NM_001278210 0.32 chr6 1.49E+08 1.49E+08 + −0.51 0.00454 2.41 3.22E−05 NM_005715 0.32 chr8 1.26E+08 1.26E+08 + −0.37 0.139459 1.73 2.08E−10 NM_005005 0.32 chr11 93868890 93869064 + −0.87 0.178044 2.24 9.42E−06 NM_015368 0.31 chr13 43996261 43996733 + 0.46 0.197118 1.55 1.36E−06 NM_001127615 0.31 chr13 44146720 44147097 + 2.52 0.000366 3.05 2.29E−13 NM_001127615 0.31 chr13 44205448 44205587 + −0.13 0.422029 4.27 6.94E−06 NM_001127615 0.31 chr4  5180013  5180217 + 0.04 0.487929 2.24 9.42E−06 NM_018401 0.31 chrX 50388208 50388386 + 1.57 0.041179 2.93 2.01E−05 NM_020717 0.31 chr12 43376666 43376912 + −1.28 0.107269 2.39 1.31E−06 NM_153026 0.3 chr17  950547  950858 + 0.74 0.054754 1.97 5.77E−08 NM_001256847 0.3 chr18 24365392 24365527 + 0.01 0.509 1.8 6.58E−05 NM_004028 0.3 chr6 26022824 26022952 + 1.93 0.000128 2.81 5.99E−05 NM_003538 0.3 chr9 82310786 82311067 + 0.72 0.192023 1.98 7.12E−06 NM_007005 0.3 chr1 43998889 43999193 + 0.68 0.182687 2.11 1.12E−06 NM_002840 0.29 chr1 2.21E+08 2.21E+08 + −0.13 0.39179 1.85 5.28E−07 NM_022746 0.29 chr11 68150884 68151023 + 1.04 0.114476 2.83 3.17E−06 NM_002335 0.29 chr12 77625139 77625382 + −0.22 0.38435 3.81 1.18E−10 NM_203394 0.29 chr2  7077412  7077553 + 0 0.593079 2.41 3.22E−05 NR_033997 0.29 chr2 1.92E+08 1.92E+08 + −0.36 0.928622 2.13 6.02E−07 NM_001254738 0.29 chr3 20659943 20660111 + 3.28 0.002807 2.2 1.53E−05 NM_001199251 0.29 chr3 20679118 20679385 + 2.26 0.010657 2.65 2.30E−07 NM_001199251 0.29 chr3 20689915 20690075 + 0.46 0.404925 3.52 4.18E−06 NM_001199251 0.29 chr3 20711116 20712204 + 1.9 1.66E−07 2.22 1.80E−20 NM_001199251 0.29 chr3 20784774 20784902 + 0.81 0.93262 4.27 6.94E−06 NM_001199251 0.29 chr3 20818080 20818222 + 0.23 0.02581 2.73 9.25E−06 NM_001199251 0.29 chr3  1.7E+08  1.7E+08 + −0.99 0.039705 1.85 9.30E−05 NM_001248008 0.29 chrX 33109524 33110058 + 0.21 0.336355 1.34 3.23E−08 NM_004010 0.29 chrX  1.4E+08  1.4E+08 + 2.34 1.66E−07 2.29 2.74E−09 NR_037183 0.29 chr10 1.34E+08 1.34E+08 + −0.54 0.22977 1.9 1.26E−06 NM_005539 0.28 chr8 20041831 20042126 + 0.92 0.063595 1.39 4.86E−05 NM_003053 0.28 chr8 1.31E+08 1.31E+08 + 1.96 5.10E−09 1.52 5.75E−09 NR_045386 0.28 chrX 39719416 39719681 + −0.54 0.3033 2.17 1.15E−07 NM_001123385 0.28 chr1 2.04E+08 2.04E+08 + 1.95 3.75E−05 1.36 9.08E−07 NM_002646 0.27 chr11 73229622 73229764 + 3.16 0.004461 3.52 4.13E−06 NM_015159 0.27 chr11 73246550 73248704 + −0.35 0.243715 2.29 2.24E−05 NM_015159 0.27 chr17 17614363 17614539 + 0.68 0.126482 2.24 9.42E−06 NM_030665 0.27 chr17 17628702 17628834 + 0.87 0.110781 3.1 3.83E−06 NM_030665 0.27 chr14 90877225 90877483 + 0.46 0.158627 3.58 2.32E−11 NM_006888 0.26 chr15 30178079 30178257 + 0.37 0.283356 3.05 2.06E−07 NM_175610 0.26 chr18  2223598  2223968 + 0.53 0.18278 1.78 2.17E−10 NM_022840 0.26 chr8 23896972 23897222 + −0.68 0.134838 2.1 4.26E−06 NM_003155 0.26 chr9 91991923 91992057 + 0.46 0.463277 3.52 4.18E−06 NM_024077 0.26 chr10 88616192 88616869 + 0.08 0.42904 1.78 4.41E−09 NM_004329 0.25 chr11 63825957 63826106 + −1.39 0.038282 2.83 3.178−06 NM_013280 0.25 chr20 51670842 51670979 + −1.28 0.039667 2.64 2.45E−06 NM_173485 0.25 chr19  5310392  5310591 + −0.35 0.243715 2.03 1.09E−05 NM_002850 0.24 chr19 14360631 14360841 + −0.17 0.344028 1.38 7.74E−05 NM_001008701 0.24 chr20 51768821 51769012 + 0.16 0.427697 1.74 6.40E−05 NM_001193421 0.24 chr21 35350428 35350593 + 0.04 0.492338 2.63 2.67E−05 NR_038883 0.24 chr5 73727131 73727292 + 0.85 0.081853 2.01 4.17E−05 NM_003633 0.24 chr5 73930549 73932593 + 1.65 1.36E−11 1.52 8.71E−26 NM_003633 0.24 chr6 48056428 48056616 + 1.53 0.01294 2.15 9.64E−05 NM_001013732 0.24 chr1 24335543 24338292 + 0.72 0.097279 1.4 2.80E−07 NR_034035 0.23 chr10 1.25E+08 1.25E+08 + 2.26 0.065139 2.73 9.25E−06 NM_004725 0.23 chr2 2.32E+08 2.32E+08 + 0.19 0.465306 3.27 4.18E−05 NR_034059 0.23 chr20 50619904 50620074 + −0.54 0.329466 4.16 1.02E−09 NM_199427 0.23 chr20 50907812 50907989 + 1.04 0.325105 2.11 3.97E−05 NM_199427 0.23 chr20 51078245 51078510 + 0.19 0.465306 1.63 9.73E−06 NM_199427 0.23 chr20 51115117 51117337 + 1.44 3.81E−09 1.43 1.41E−29 NM_199427 0.23 chr3 1.54E+08 1.54E+08 + −1.36 0.791388 2.01 4.17E−05 NM_001251962 0.23 chr1  954406  954593 + −0.22 0.35557 2.38 8.30E−06 NM_198576 0.22 chr1 2.06E+08 2.06E+08 + 0.78 0.077498 1.82 1.95E−06 NM_001123168 0.22 chr11 16932012 16932827 + 1.29 0.006935 1.91 5.30E−13 NM_175058 0.22 chr16 73948502 73948683 + 3.36 8.86E−06 1.37 0.00236 NM_002811 0.22 chr17  2137082  2137379 + 0.71 0.070162 1.34 2.25E−05 NM_001256828 0.22 chr19 17202119 17202422 + 0.16 0.32225 2.04 1.48E−08 NM_001130065 0.22 chr5 56136138 56136266 + 0.46 0.404925 3.58 2.33E−06 NM_005921 0.22 chr8 1.24E+08 1.24E+08 + 1.78 0.149771 1.34 1.42E−05 NM_024295 0.22 chr8 1.42E+08 1.42E+08 + 0.71 0.005577 1.94 1.22E−12 NM_001164823 0.22 chr1 43941605 43941830 + −1.13 0.188905 1.82 1.95E−06 NM_001190880 0.21 chr1 1.58E+08 1.58E+08 + 1.92 0.035602 2.31 8.78E−05 NM_018240 0.21 chr1 1.58E+08 1.58E+08 + 0.18 0.360591 1.72 3.20E−07 NM_018240 0.21 chr13 91369397 91369807 + 0.64 0.115893 1.44 2.32E−05 NR_027039 0.21 chr17 45982250 45982748 + 1.26 0.011922 1.46 6.31E−06 NM_003110 0.21 chr4  1.6E+08  1.6E+08 + 1.63 0.016149 2.3 9.22E−07 NM_014247 0.21 chr4 1.61E+08 1.61E+08 + 2.46 0.042327 3.34 2.36E−05 NM_014247 0.21 chr5 1.72E+08 1.72E+08 + 0.78 0.25643 1.46 1.58E−06 NM_001017995 0.21 chr9 1.07E+08 1.07E+08 + 0.9 0.001929 1.37 4.39E−05 NM_001042551 0.21 chr9 1.07E+08 1.07E+08 + 0.87 0.210905 3.2 1.25E−06 NM_001042551 0.21 chr9 1.07E+08 1.07E+08 + 1.36 0.044821 1.46 4.04E−05 NM_001042550 0.21 chr9 1.07E+08 1.07E+08 + 1.16 0.08465 3.25 7.10E−07 NM_001042550 0.21 chr1 58875361 58876232 + 0.68 0.090078 1.45 9.60E−09 NM_145243 0.2 chr15 57550260 27550529 + 1.68 0.006504 1.95 1.12E−05 NM_207040 0.2 chr17  2511784  2513068 + 1.42 1.22E−05 1.39 1.40E−12 NM_000430 0.2 chr17 20932205 20932373 + 2.1 0.001188 2.41 3.22E−05 NM_015276 0.2 chr18 56589949 56590098 + −0.41 0.166483 3.15 2.19E−06 NM_018181 0.2 chr11 64611591 64611765 + −1.03 0.107039 2.68 1.57E−05 NM_017525 0.19 chr15 68060682 58060979 + 0.04 0.504309 1.46 4.04E−05 NM_001258024 0.19 chr17  1286044  1286197 + 0.26 0.418658 1.68 6.15E−05 NM_006761 0.19 chr17 28244948 28245077 + 1.04 0.325105 2.81 5.99E−05 NM_001145053 0.19 chr19 34050055 34050355 + −0.54 0.3033 1.44 3.65E−06 NM_000285 0.19 chr6 1.35E+08 1.35E+08 + 0.46 0.308555 2.31 1.69E−09 NR_038219 0.19 chr9 1.23E+08 1.23E+08 + 0.13 0.464479 2.58 4.50E−05 NR_073557 0.19 chr1 27891918 27892088 + 0.9 0.083608 3.12 6.79E−08 NM_001029882 0.18 chr14 59788968 59789229 + 1.11 0.036985 3.54 1.28E−08 NM_014992 0.18 chr18 47325795 47325925 + 1.57 0.041179 2.46 1.94E−05 NM_006111 0.18 chr20 56236365 56236537 + 1.46 0.00118 2.36 5.33E−05 NM_001255976 0.18 chr20 56241983 56242159 + 4.21 5.50E−06 1.6 0.00054 NM_001255976 0.18 chr20 62091442 62091654 + 0.51 0.155661 1.8 6.58E−05 NM_172107 0.18 chr20 62093991 62094204 + 1.11 0.036985 2.75 4.74E−08 NM_172107 0.18 chr4 1.24E+08 1.24E+08 + 1.9 1.16E−18 2.24 1.03E−23 NM_001258038 0.18 chr4 1.24E+08 1.24E+08 + 2.08 4.82E−20 1.42 1.23E−07 NM_001258038 0.18 chr9 45045301 45045495 + 0.46 0.334119 1.8 6.58E−05 NR_027421 0.18 chrX  1.3E+08  1.3E+08 + 2.12 0.004281 2.01 4.17E−05 NR_027141 0.18 chrX  1.3E+08  1.3E+08 + 2.98 0.000217 1.64 2.60E−06 NR_027141 0.18 chrX  1.3E+08  1.3E+08 + 1.6 3.06E−11 1.95 5.51E−33 NR_027141 0.18 chrX  1.3E+08  1.3E+08 + 0.04 0.492338 1.42 5.28E−06 NR_027141 0.18 chr1 16179816 16179944 + 0.04 0.504309 3.1 3.83E−06 NR_024279 0.17 chr10 31261588 31251817 + −0.34 0.258417 1.84 1.76E−05 NM_001143769 0.17 chr12 1.05E+08 1.05E+08 + −0.54 0.281197 3.4 1.33E−05 NM_018413 0.17 chr12 1.05E+08 1.05E+08 + −0.22 0.38435 1.81 1.12E−05 NM_018413 0.17 chr16  2232770  2232941 + −2.54 0.044242 3.1 3.83E−06 NM_020764 0.17 chr2 12559899 12560135 + 0.23 0.317498 1.58 3.45E−05 NM_021643 0.17 chr20 59860902 59861030 + −0.54 0.245002 2.93 2.01E−05 NM_001794 0.17 chr20 60136453 60136623 + −1.13 0.065395 1.91 1.75E−05 NM_001252339 0.17 chr4 56964871 56965203 + −0.22 0.35557 1.65 9.30E−05 NM_020722 0.17 chr1  1.5E+08  1.5E+08 + 0.36 0.303275 3.27 4.18E−05 NM_001136479 0.16 chr11 46959447 46959748 + 0.97 0.064254 1.51 1.88E−05 NM_001003678 0.16 chr15 59018593 59018761 + 2.26 0.065139 3.68 7.23E−07 NM_001110 0.16 chr15 80711486 80711619 + 2.16 0.016043 2.81 5.99E−05 NM_014862 0.16 chr17  631552  631722 + 0.19 0.465306 1.8 6.58E−05 NM_024792 0.16 chr18 60460282 60460410 + 0.46 0.308555 2.78 5.42E−06 NM_194449 0.16 chr18 60468692 60468831 + −0.71 0.148471 2.07 6.37E−05 NM_194449 0.16 chr2 98222338 98222803 + 1.46 0.018075 1.5 1.86E−06 NM_025190 0.16 chr5 15069959 15070110 + 1.78 0.052385 2.68 1.57E−05 NM_054027 0.16 chr1  7434513  7434731 + 2.03 1.02E−05 1.4 0.00035 NM_004781 0.15 chr11 88027196 88027324 + 0.81 0.005384 3.05 6.68E−06 NM_001814 0.15 chr11 89981373 89981533 + −2.42 0.002298 2.31 8.78E−05 NM_001144073 0.15 chr19 49116753 49116978 + 0.75 0.100892 1.9 1.26E−06 NM_0177708 0.15 chr19 49591785 49591916 + 1.38 0.025068 2.58 4.50E−05 NM_003089 0.15 chr20 40021741 40021872 + −1.87 0.054231 3.63 1.30E−08 NM_052846 0.15 chr3 1.28E+08 1.28E+08 + 0.04 0.529972 2.36 5.33E−05 NM_207335 0.15 chr5 67702765 67702894 + −1.22 0.064934 3.34 2.36E−05 NM_001242466 0.15 chr8 26059915 26060074 + 2.63 0.027294 2.51 1.16E−05 NM_002717 0.15 chr9  2414499  2414664 + 0.19 0.465306 2.78 5.42E−06 NM_003383 0.15 chrX 46455295 46455502 + 1.68 0.002665 1.88 2.74E−05 NM_019886 0.15 chr16 67598952 67599211 + 0.37 0.283356 1.46 2.53E−05 NM_006565 0.14 chr2 64913142 64913309 + 0.46 0.365076 2.92 1.07E−06 NM_014755 0.14 chr20 25605631 25605770 + 1.36 0.044821 1.84 1.75E−05 NM_152667 0.14 chr3 39069341 39069929 + 1.26 0.000269 1.89 2.30E−13 NM_020839 0.14 chr6 1.51E+08 1.51E+08 + −0.96 0.004213 1.82 3.38E−10 NM_030949 0.14 chr1 21491558 21491724 + 1.04 0.156144 2.1 4.28E−06 NM_001198803 0.13 chr1 87584227 87584355 + 1.68 0.028759 2.88 3.48E−05 NR_026989 0.13 chr1 1.81E+08 1.81E+08 + 0.11 0.462988 1.44 1.46E−05 NM_005819 0.13 chr1 2.43E+08 2.43E+08 + 0.26 0.418658 3.09 1.18E−07 NR_029401 0.13 chr1 2.43E+08 2.43E+08 + 1.63 0.07633 2.01 4.17E−05 NR_029401 0.13 chr12 77075107 77075235 + 1.26 0.156387 3.46 7.46E−06 NM+015336 0.13 chr19 48162101 48162258 + 0.54 0.204578 1.96 6.59E−05 NM_015711 0.13 chr22 37994944 37995624 + 1.13 0.083706 1.51 5.97E−10 NM_013365 0.13 chr5 1.53E+08 1.53E+08 + 0.87 0.062557 2.16 2.45E−17 NM_001258022 0.13 chr5 1.53E+08 1.53E+08 + 1.46 0.109973 3.63 1.30E−06 NM_001258022 0.13 chr5 1.53E+08 1.53E+08 + 0.76 3.05E−06 1.34 1.63E−18 NM_001258022 0.13 chr5 1.53E+08 1.53E+08 + 2.7 5.57E−06 2.2 1.32E−13 NM_001258022 0.13 chr5 1.53E+08 1.53E+08 + 0.98 0.049001 3.05 3.33E−22 NM_001258022 0.13 chr5 1.53E+08 1.53E+08 + −0.36 0.926622 4.27 6.94E−06 NM_001258022 0.13 chr5 1.53E+08 1.53E+08 + 1.06 0.000284 1.45 2.16E−15 NM_001258022 0.13 chr5 1.53E+08 1.53E+08 + 3.92 6.26E−05 3 2.07E−11 NM_001258022 0.13 chr5 1.53E+08 1.53E+08 + 2.4 7.49E−13 1.99 3.90E−27 NM_001258022 0.13 chr5 1.53E+08 1.53E+08 + −0.54 0.329466 4.34 3.77E−06 NM_001258022 0.13 chr5 1.53E+08 1.53E+08 + −0.96 0.18457 3.68 7.23E−07 NM_001258022 0.13 chr6 87921887 87922118 + −0.18 0.381896 1.95 1.12E−05 NM_015021 0.13 chr6 1.66E+08 1.66E+08 + 0.57 0.230388 1.36 6.99E−05 NM_144980 0.13 chr1 21446824 21447079 + 0.78 0.077498 1.44 2.32E−05 NM_003760 0.12 chr15 81217868 81218519 + −0.75 0.044073 2.01 5.42E−11 NM_015154 0.12 chr16 89539456 89539585 + 0.26 0.418658 2.15 9.64E−05 NM_013275 0.12 chr18 42268830 42268964 + 0.46 0.334119 1.74 6.40E−05 NM_015559 0.12 chr19 39356017 39356256 + −0.96 0.104912 1.7 9.76E−05 NM_198445 0.12 chr19 54613297 54613451 + 0.46 0.158627 2.2 1.53E−05 NM_015629 0.12 chr22 36341267 36341395 + −0.32 0.326824 4.4 2.04E−06 NM_001082578 0.12 chr3 55356599 55358782 + 1.46 0.109973 2.03 1.19E−06 NM_001256105 0.12 chr3 55356599 55358782 + 1.04 0.003074 1.8 4.63E−17 NM_001256105 0.12 chr3 55457392 55457817 + 0.46 0.176457 1.61 6.61E−07 NM_001256105 0.12 chr3 55458323 55459016 + 0.36 0/231846 4.76 1.30E−35 NM_001256105 0.12 chr3 55460115 55462031 + 0.57 0.034711 1.94 1.18E−29 NM_001256105 0.12 chr3 55500856 55501335 + 0.68 0.280156 1.63 7.60E−08 NM_001256105 0.12 chr5 1.53E+08 1.53E+08 + −1.35 0.06335 1.84 6.01E−08 NM_005927 0.12 chr7 1.54E+08 1.54E+08 + 0.81 0.93262 2.63 2.67E−05 NR_024477 0.12 chr9  7983611  7983880 + 0.29 0.337406 1.52 7.61E−05 NM_033428 0.12 chr9 27086846 27087018 + −0.78 0.049269 1.96 6.59E−05 NM_000459 0.12 chr11 1.11E+08 1.11E+08 + 0.96 0.084864 1.31 1.25E−07 NR_034154 0.11 chr11 1.11E+08 1.11E+08 + 1.34 0.000868 1.95 9.04E−08 NR_034154 0.11 chr11 1.11E+08 1.11E+08 + 0.64 0.115893 1.34 1.42E−05 NR_034154 0.11 chr11 1.11E+08 1.11E+08 + 0.56 0.077732 1.68 1.67E−12 NR_034154 0.11 chr17  3570198  3570450 + 1.1 0.028425 1.64 1.54E−05 NM_014604 0.11 chr2  1.1E+08  1.1E+08 + 1.04 0.06437 1.7 2.56E−05 NM_144710 0.11 chr2  1.1E+08  1.1E+08 + 0.01 0.504539 1.87 4.60E−17 NM_144710 0.11 chr20 47385126 47385418 + 1.13 0.010049 1.53 3.19E−06 NM_020820 0.11 chr4 88437065 88437918 + 0.33 0.119205 1.4 4.08E−10 NM_004684 0.11 chr5 88172041 88172169 + 1.04 0.325105 2.26 1.64E−07 NM_001193350 0.11 chr9 45609849 45610441 + −0.54 0.19189 1.46 2.51E−06 NR_024060 0.11 chr9 1.28E+08 1.28E+08 + 0.46 0.404925 2.16 2.47E−05 NM_001006617 0.11 chr1 14117179 14117511 + 0.65 0.083141 1.44 1.46E−05 NM_001007257 0.1 chr10 82241644 82242201 + 1.7 0.000463 1.3 8.05E−05 NM_001128309 0.1 chr11 63914872 63915402 + −0.25 0.332095 1.9 2.20E−07 NM_014067 0.1 chr18 52891566 52891861 + 0.67 0.062848 1.44 9.18E−06 NM_001243236 0.1 chr18 52997517 52997653 + 0.93 0.048668 2.01 4.17E−05 NM_001243235 0.1 chr18 53028093 53028270 + 1.46 0.032155 1.97 2.96E−06 NM_001243235 0.1 chr18 53199411 53199573 + 0.28 0.418658 2.38 8.30E−06 NM_001243231 0.1 chr2 11594473 11594661 + 4.21 5.50E−06 1.68 0.000236 NR_003095 0.1 chr5 1.57E +08 1.57E+08 + 0.19 0.356801 1.85 3.03E−06 NM_033274 0.1 chr9 1.27E+08 1.27E+08 + −1.39 0.006059 2.46 1.94E−05 NM_002799 0.1 chr9 1.345+08 1.34E+08 + 1.46 0.018075 1.88 1.43E−07 NR_033701 0.1 chr12 22925635 22925784 + 2.26 0.065139 2.15 9.64E−05 NM_0186385 0.09 chr15 41406286 41406414 + 0.78 0.201565 2.06 1.15E−06 NM_017553 0.09 chr16 85598720 85599221 + 1.26 1.16E−05 1.51 2.03E−07 NM_001134473 0.09 chr18  693503  693735 + 0.57 0.250388 1.73 1.68E−05 NM_202758 0.09 chr3 1.25E+08 1.25E+08 + 1.17 0.027729 1.61 2.315−05 NM_003794 0.09 chr9  1.1E+08  1.1E+08 + 2.4 0.000845 2.31 8.78E−05 NM_001244713 0.09 chr1 38462438 38482676 + 2.92 1.15E−05 2.12 1.03E−05 NM_006802 0.08 chr12 1.24E+08 1.24E+08 + 0.87 0.000738 1.37 6.27E−06 NM_001270434 0.08 chr17 17387773 17387901 + 1.46 0.032155 2.63 2.67E−05 NM_001270434 0.08 chr18  3360281  3360622 + −0.31 0.198324 1.43 3.36E−07 NM_174886 0.08 chr2 56029291 56030156 + 0.3 0.192847 1.35 2.41E−09 NM_033109 0.08 chr2 56032718 56033331 + 2.12 4.01E−10 1.38 2.26E−09 NM_033109 0.08 chr4 61689874 61690013 + −1.13 0.188905 3.46 7.46E−06 NM_015236 0.08 chr4 80555530 80555666 + 2.36 0.007037 2.07 6.37E−05 NM_038826 0.08 chr9 1.39E+08 1.39E+08 + −0.46 0.166558 2.83 3.17E−08 NM_001080849 0.08 chr1 1.81E+08 1.81E+08 + 0.53 0.18278 1.76 7.78E−10 NM_032360 0.07 chr1 2.03E+08 2.03E+08 + 0.54 0.204578 1.32 4.59E−06 NM_001197131 0.07 chr1 2.32E+08 2.32E+08 + −0.23 0.252632 2.43 1.26E−07 NM_001164549 0.07 chr10 97099932 97100073 + −1.54 0.060085 2.01 4.17E−05 NM_020992 0.07 chr11 36262179 36262783 + −1.01 0.006775 1.59 1.85E−08 NM_001101653 0.07 chr11 67157991 67158339 + 2.46 0.000557 1.48 1.09E−05 NM_004584 0.07 chr3 39002898 39003141 + 1.04 0.017239 2.65 2.30E−07 NM_014139 0.07 chr5 32056159 32056672 + 0.02 0.486295 1.44 5.81E−07 NM_022130 0.07 chr5 32145928 32146372 + 1.13 0.012899 1.34 6.30E−05 NM_022130 0.07 chr7  5481662  5481790 + 0.82 0.10778 1.74 6.40E−05 NM_001080495 0.07 chr9  1.2E+08  1.2E+08 + −0.22 0.38435 1.85 7.28E−06 NM_012210 0.07 chr9  1.2E+08  1.2E+08 + 2.03 1.02E−05 2.25 0.000144 NM_012210 0.07 chr9 1.26E+08 1.26E+08 + 0.19 0.465306 1.6 8.76E−05 NM_026677 0.07 chr13 33219125 33219385 + −0.01 0.470937 1.41 8.54E−05 NM_015032 0.06 chr19 55914629 55914903 + 1.96 0.009351 1.67 1.02E−05 NM_014501 0.06 chr2 54226039 54226282 + −1.03 0.107039 2.09 4.71E−07 NM_014814 0.06 chr21 35199006 35199187 + 0.57 0.230388 2.68 1.57E−05 NM_001697 0.06 chr6 34781221 34781593 + −0.77 0.163179 1.31 5.07E−05 NM_017754 0.06 chr7 44796472 44796875 + 0.52 0.164068 1.42 8.38E−06 NM_174929 0.06 chr17 47516437 47517063 + 1.95 3.75E−05 1.36 4.39E−10 NM_002634 0.05 chr17 47518750 47520003 + 2.86 7.79E−11 2.1 9.77E−25 NM_002634 0.05 chr17 47521248 47521451 + 0.46 0.334119 2.46 3.03E−06 NM_002634 0.05 chr17 47530054 47530228 + 1.92 0.003755 2.5 1.82E−06 NM_002634 0.05 chr2 39950736 39951042 + 0.74 0.041562 1.77 2.16E−07 NR_028102 0.05 chr2 2.17E+08 2.17E+08 + −0.46 0.175792 1.7 1.81E−06 NM_000597 0.05 chr20 48986691 48986940 + 1.26 0.011922 2.5 1.82E−06 NR_034124 0.05 chr4 1.84E+08 1.84E+08 + 2.04 0.099329 1.7 9.76E−05 NM_001921 0.05 chr5 1.54E+08 1.54E+08 + −0.54 0.203416 1.91 1.75E−05 NM_015315 0.05 chr9 1.24E+08 1.24E+08 + 0.11 0.482988 1.7 9.76E−05 NM_016322 0.05 chr19 18546658 18546851 + 1.41 0.004736 1.98 7.12E−06 NM_001253389 0.04 chr2  2.1E+08  2.1E+08 + 0.36 0.231846 1.85 7.28E−08 NM_001039538 0.04 chr5 1.53E+08 1.53E+08 + 0.84 0.148331 1.43 5.89E−05 NM_000827 0.04 chr7 1.51E+08 1.51E+08 + 1.42 3.40E−05 2.04 5.04E−22 NM_005614 0.04 chr9  2812940  2813110 + 0.87 0.110781 2.24 9.42E−06 NM_014878 0.04 chr1 1.04E+08 1.04E+08 + 1.48 6.30E−05 1.64 1.99E−14 NM_080629 0.03 chr1 1.04E+08 1.04E+08 + 2.09 2.83E−09 1.86 3.02E−12 NM_080629 0.03 chr1 1.68E+08 1.68E+08 + 0.57 0.230388 2.49 7.40E−09 NM_001146191 0.03 chr1 2.05E+08 2.05E+08 + 0.45 0.015307 2.52 7.56E−05 NM_001242925 0.03 chr17 61330184 61330440 + 1.54 0.022876 1.72 4.05E−05 NM_001017917 0.03 chr19  3609028  3609939 + 0.6 0.011601 1.61 1.33E−12 NR_038865 0.03 chr19 50531358 50531748 + 2.63 0.001965 1.85 2.21E−07 NM_015428 0.03 chr2 56114610 46115390 + 1.53 0.000621 1.36 5.99E−11 NM_001039349 0.03 chr2 56115899 56116414 + 1.59 0.000322 1.43 2.13E−07 NM_001039349 0.03 chr2 68474090 68474315 + 0.13 0.464479 2.46 3.03E−06 NM_000945 0.03 chr2  2.1E+08  2.5E+08 + 1.3 0.0345 4.27 6.94E−06 NM_031847 0.03 chr2 2.38E+08 2.38E+08 + 0.23 0.02581 3.34 2.36E−05 NM_198189 0.03 chr2 2.38E+08 2.38E+08 + 0.01 0.509 1.81 2.71E−05 NM_198189 0.03 chr22 30350374 30350528 + −0.54 0.261994 3.34 2.36E−05 NM_153050 0.03 chr3 1.44E+08 1.44E+08 + −0.3 0.29184 1.49 4.40E−05 NM_001134470 0.03 chr3 1.71E+08 1.71E+08 + 0.04 0.504309 1.96 6.59E−05 NM_001161562 0.03 chr3 1.71E+09 1.71E+08 + 0.78 0.044012 1.54 2.02E−05 NM_001161562 0.03 chr6 1.57E+08 1.57E+08 + −1.13 0.188905 1.66 6.47E−06 NM_018452 0.03 chr7  6290528  6290738 + 1.22 0.047091 2.03 1.19E−06 NM_004227 0.03 chr7  6308678  6308661 + 2.19 0.002873 1.95 2.71E−05 NM_004227 0.03 chr7 1.02E+08 1.02E+08 + −1.28 0.107269 1.79 7.19E−06 NM_181552 0.03 chr10 1.24E+08 1.24E+08 + 1.63 0.07633 2.42 5.02E−06 NM_007041 0.02 chr13 25706143 25706271 + 0 0.593079 3.46 7.48E−06 NM_030979 0.02 chr17  7729203  7729331 + 0 0.593079 4.27 6.94E−06 NM_001080424 0.02 chr19 47413985 47414334 + 1.33 0.082052 1.54 1.27E−05 NM_004491 0.02 chr6 68944702 68944830 + 1.46 0.222672 4.27 6.94E−06 NM_001704 0.02 chr7 73919392 73919699 + 1.11 0.036985 2.46 4.70E−07 NM_005685 0.02 chr7 1.41E+08 1.41E+08 + 1.92 0.035602 2.78 5.42E−06 NM_018238 0.02 chr9   1E+08   1E+08 + 0.54 0.111688 1.3 1.81E−05 NM_002486 0.02 chr1 43672570 43672957 + −0.03 0.460616 1.31 5.07E−05 NM_001159936 0.01 chr1 1.56E+08 1.56E+08 + 1.96 0.009351 2.2 5.95E−05 NM_004632 0.01 chr10 61316968 61317858 + 2.32 6.88E−09 2.06 4.02E−21 NM_194298 0.01 chr12 1.25E+08 1.25E+08 + −0.1 0.415961 3.34 2.30E−05 NM_001082959 0.01 chr12 1.25E+08 1.25E+08 + 1.78 0.149771 2.88 3.48E−05 NM_001082959 0.01 chr20 60622265 60622463 + −0.16 0.376068 1.52 7.61E−05 NM_003185 0.01 chr3 1.49E+08 1.49E+08 + 0.92 0.063595 1.88 6.65E−05 NM_001188280 0.01 chr4  3353361  3353573 + 1.36 0.044821 1.43 9.39E−05 NM_188227 0.01 chr5 1.42E+08 1.42E+08 + 0.61 0.00457 1.98 1.33E−10 NM_001127496 0.01 chr7 75963781 75063983 + 0.92 0.152933 2.01 4.17E−05 NM_012479 0.01 chr1 32580306 32580449 + −0.28 0.353275 3.05 6.68E−06 NM_012316 0 chr1 59275435 59275615 + 0.26 0.418658 2.41 3.22E−05 NR_034015 0 chr1 2.14E+08 2.14E+08 + 1.4 0.014239 1.71 1.79E−12 NR_046189 0 chr1 2.14E+08 2.14E+08 + 2.16 984E−09 1.5 3.44E−15 NR_046189 0 chr1 2.34E+08 2.34E+08 + 1.18 0.000393 1.43 8.94E−13 NR_039625 0 chr1 2.48E+08 2.48E+08 + 0.52 0.173082 1.67 2.51E−09 NM_001004698 0 chr10 10656127 10656264 + 1.46 0.222672 2.25 3.66E−05 NR_027082 0 chr10 24595354 24595567 + 1.46 0.109973 2.16 2.47E−05 NR_030334 0 chr10 31064331 31065108 + −0.36 0.136534 1.33 5.35E−09 NM_183058 0 chr10 45676458 45877069 + 2.23 1.18E−12 1.52 1.42E−08 NR_036112 0 chr10 50012899 50013139 + 0.46 0.404925 1.78 1.73E−05 NM_001006939 0 chr10 63554022 63554150 + 0.23 0.972582 4.58 3.23E−07 NM_032199 0 chr10 80636627 80636995 + 2.46 0.004621 1.49 1.735−05 NR_015429 0 chr10   1E+08   1E+08 + −0.54 0.261994 2.06 7.49E−07 NR_031619 0 chr10   1E+08   1E+08 + 2.93 1.73E−41 2.84 7.33E−54 NR_031619 0 chr10   1E+08   1E+08 + 4.1 6.48E−10 4.22 7.66E−19 NR_031619 0 chr10   1E+08   1E+08 + 1.84 2.03E−05 1.72 1.40E−13 NR_031619 0 chr10 1.26E+08 1.26E+08 + 0 0.593079 2.28 5.78E−08 NM_001146340 0 chr10 1.32E+08 1.32E+08 + 1.92 0.035602 2.583 1.73E−07 NR_036179 0 chr10 1.35E+08 1.35E+08 + −0.36 0.926622 2.15 9.64E−05 NR_030170 0 chr11 27641447 27641638 + 2.16 0.016043 2.38 8.30E−06 NR_102708 0 chr11 57792244 57792419 + 0.45 0.015307 4.78 2.72E−08 NM_001005212 0 chr11 57825789 57826451 + 0.4 0.14411 1.36 2.93E−09 NM_001005186 0 chr11 63645918 63646056 + 3.46 0.0011 3.34 2.36E−05 NM_017490 0 chr11 64173804 64173981 + −0.36 0.926622 3.25 7.10E−07 NR_031602 0 chr11 79115084 79115212 + −0.54 0.36033 2.93 2.01E−05 NR_030598 0 chr11 1.11E+08 1.11E+08 + 1.32 2.63E−05 1.43 1.35E−20 NR_039712 0 chr11 1.18E+08 1.18E+08 + −1.54 0.103108 2.93 2.01E−05 NM_001558 0 chr11 1.27E+08 1.27E+08 + −0.01 0.473517 1.78 3.84E−08 NR_046986 0 chr11 1.27E+08 1.27E+08 + −0.17 0.344028 1.38 1.08E−09 NR_040078 0 chr11 1.27E+08 1.27E+08 + 0.46 0.308555 1.76 1.10E−05 NR_040078 0 chr11 1.31E+08 1.31E+08 + 0.46 0.308555 1.66 1.73E−06 NM_001271983 0 chr11 1.35E+08 1.35E+08 + 1.1 0.001894 2.63 8.53E−16 NR_033852 0 chr11 1.35E+08 1.35E+08 + 0.41 0.197649 3.49 5.48E−31 NR_033852 0 chr11 1.35E+08 1.35E+08 + 2.3 2.10E−09 3.14 5.38E−29 NR_033852 0 chr11 1.35E+08 1.35E+08 + 2.1 6.05E−06 3.38 1.29E−07 NR_033852 0 chr11 1.35E+08 1.35E+08 + 1.46 0.032155 3.92 6.18E−15 NR_033852 0 chr11 1.35E+08 1.35E+08 + 3.13 1.79E−08 4.16 7.70E−43 NR_033852 0 chr11 1.35E+08 1.35E+08 + 3.36 2.77E−47 2.36 7.99E−54 NR_033852 0 chr11 1.35E+08 1.35E+08 + 3.94 1.53E−12 2.58 6.51E−07 NR_033852 0 chr11 1.35E+08 1.35E+08 + 1.19 0.113922 2.38 6.30E−11 NR_033852 0 chr12 11969210 11969339 + 0.09 0.461298 3.1 3.83E−06 NR_046487 0 chr12 13628016 13628202 + 0.04 0.513686 1.56 8.19E−05 NR_036555 0 chr12 13761072 13761200 + 3.46 0.0011 4.2 1.27E−05 NR_036555 0 chr12 14994804 14994960 + −0.13 0.422029 2.99 1.16E−05 NM_021071 0 chr12 25877258 25877388 + 0.78 0.201565 2.29 2.24E−05 NM_001145727 0 chr12 43681762 43682309 + 2.23 2.05E−05 2.78 8.39E−13 NM_025003 0 chr12 43683291 43683681 + 1.08 0.010278 2.89 3.40E−14 NM_025003 0 chr12 81601985 81602204 + 0.23 0.02581 2.11 3.97E−05 NR_039848 0 chr12 97651518 97651694 + −0.81 0.170714 3.68 7.23E−07 NR_024037 0 chr12 98479564 96480254 + 3.16 1.14E−08 2.35 2.44E−15 NR_036189 0 chr12 1.23E+08 1.23E+08 + 0.65 0.001184 1.35 2.80E−08 NM_024667 0 chr12 1.23E+08 1.23E+08 + 0.45 0.015307 2.92 1.07E−06 NM_001002251 0 chr13 26538091 26538326 + 0.82 0.10778 1.46 6.44E−05 NM_001007538 0 chr13 33396887 33397116 + 0.39 0.249424 1.6 8.76E−05 NR_047020 0 chr13 40519502 40519690 + 0.46 0.404925 2.2 5.95E−05 NR_046870 0 chr13 43198084 43198362 + 0.81 0.005384 2.1 4.28E−06 NM_003701 0 chr13 43325163 43325336 + 0.81 0.005384 2.41 3.22E−05 NM_182508 0 chr13 46470026 46470653 + 1.19 0.113922 1.51 3.26E−08 NM_198849 0 chr13 53739670 53739798 + 0.45 0.015307 4.88 7.84E−09 NM_006418 0 chr13 53743826 53744167 + 0.19 0.465306 2.95 6.17E−11 NM_006418 0 chr13 53744968 53745622 + 1.87 0.013694 3.23 1.70E−17 NM_006418 0 chr13 53771722 53773846 + 1 0.000149 1.45 4.70E−21 NM_006418 0 chr13 78949336 78949576 + 1.55 0.000234 1.65 9.30E−05 NM_006237 0 chr13 78977631 78977802 + 0.96 0.003193 4.73 5.05E−08 NM_006237 0 chr13 79361003 79361131 + −2.36 0.648257 3.34 2.36E−05 NR_046869 0 chr14 61933119 61933261 + −0.06 0.44608 2.78 5.42E−06 NR_039985 0 chr14 85426819 85426979 + 0.45 0.015307 2.93 2.01E−05 NR_102737 0 chr14 1.05E+08 1.05E+08 + −1.13 0.025762 1.52 1.27E−06 NM_001008404 0 chr15 39218732 39218942 + 0.04 0.529972 2.64 2.45E−06 NM_207444 0 chr15 60159346 60159882 + 0.19 0.424653 1.75 3.76E−08 NM_012182 0 chr15 62757376 62757514 + −1.13 0.108085 2.92 1.07E−06 NR_026897 0 chr15 74574650 74674806 + 1.33 0.082052 2.73 9.25E−06 NM_025055 0 chr15 75982744 75982924 + 1.04 0.04895 3.1 3.83E−06 NM_001897 0 chr15 89154155 89154622 + 0.46 0.365076 2.08 5.91E−09 NR_029606 0 chr16 55779899 55780066 + −0.37 0.283497 2.07 6.37E−05 NR_003276 0 chr16 55823601 55824462 + 0.86 0.027931 1.74 1.04E−11 NR_003276 0 chr16 57841079 57841208 + 1.46 0.222672 2.93 2.01E−05 NM_001278081 0 chr16 57972671 57972899 + 2.26 0.065139 1.68 1.62E−05 NM_001297 0 chr16 73581552 73582284 + 1.64 0.000139 2.05 1.60E−12 NR_038234 0 chr16 73705089 73705396 + 0.68 0.280156 1.73 1.68E−05 NR_038234 0 chr16 85387355 85387561 + −0.35 0.30382 2.58 4.50E−05 NR_049816 0 chr17 18973077 18973205 + −0.96 0.104912 3.73 4.01E−07 NM_006613 0 chr17 30023224 30023358 + 0.04 0.529972 2.81 5.99E−05 NR_029856 0 chr17 68106484 68106709 + 1.16 0.08465 1.59 5.50E−05 NM_018658 0 chr17 71851797 71852183 + 0.68 0.126482 1.52 1.27E−06 NR_027146 0 chr17 75880302 75880439 + −0.54 0.3033 2.93 2.01E−05 NR_028337 0 chr17 77644303 77644849 + 1.5 0.002493 2.68 1.12E−13 NR_039893 0 chr18  6541217  6541352 + 0.78 0.301098 2 1.88E−06 NM_001243517 0 chr18 14912993 14913127 + −0.36 0.926622 3.38 1.29E−07 NR_036153 0 chr18 14971594 14971796 + 1.34 0.000666 2.38 5.51E−08 NR_036153 0 chr18 14972405 14972547 + 1.36 0.044821 2.68 1.57E−05 NR_036153 0 chr18 14986724 14986915 + 3.46 0.0011 2.96 6.20E−07 NR_036153 0 chr18 15067063 15067959 + 1.96 4.26E−07 1.56 4.63E−12 NR_036153 0 chr18 15070052 15070235 + 0.64 0.009079 2.73 9.25E−06 NR_036153 0 chr18 27197929 27198168 + 1.92 0.035602 1.49 7.02E−05 NR_031684 0 chr18 27292843 27292992 + 0.9 0.083608 1.88 2.74E−05 NR_031684 0 chr18 43414021 43414945 + −0.17 0.314291 1.41 1.25E−17 NM_213602 0 chr18 56155573 56155747 + −0.16 0.376068 1.8 6.58E−05 NR_039899 0 chr18 69776124 69776263 + 1.57 0.041179 3.73 4.01E−07 NM_182511 0 chr19 18401815 18401953 + −0.18 0.381896 2.46 1.94E−05 NR_036155 0 chr2 17454049 17454858 + 1.17 0.027729 3.59 1.76E−21 NM_001099218 0 chr2 21473721 21474996 + 0.65 0.083141 2.49 8.28E−26 NM_000384 0 chr2 21479032 21479219 + 1.26 0.156387 3.29 4.03E−07 NM_000384 0 chr2 21482960 21483383 + 2.26 0.010657 4.12 1.66E−13 NM_000384 0 chr2 21484000 21485005 + 4.57 5.78E−14 3.96 7.17E−33 NM_000384 0 chr2 21485602 21486129 + 2.83 2.77E−05 3.06 1.47E−16 NM_000384 0 chr2 21512539 21512779 + 0.45 0.015307 5.01 1.21E−09 NM_000384 0 chr2 21812857 21813002 + 0.78 0.301098 2.19 3.14E−07 NR_038837 0 chr2 33090422 33090693 + 0.84 0.148331 1.82 4.67E−06 NR_027099 0 chr2 56355601 56355735 + 0.96 0.003193 2.32 8.53E−08 NM_001080433 0 chr2 1.35E+08 1.35E+08 + 0.81 0.93262 2.52 7.56E−05 NR_037450 0 chr2 1.83E+08 1.83E+08 + 1.68 0.028759 2.05 2.62E−05 NM_001080545 0 chr2 1.86E+08 1.86E+08 + 1.34 0.000666 2.32 3.54E−06 NM_173651 0 chr2 2.15E+08 2.15E+08 + 1.04 0.325105 3.96 2.06E−08 MR_047698 0 chr2  2.4E+08  2.4E+08 + 0.04 0.492338 2.28 5.78E−06 NR_036229 0 chr20 49971625 49972134 + 0.9 0.083608 1.35 3.58E−06 NR_036182 0 chr20 56534294 56534456 + 0.68 0.182687 2.01 4.17E−05 NR_039757 0 chr21 41425428 41425556 + −0.36 0.926622 3.2 7.38E−05 NR_039917 0 chr21 41429262 41429575 + 2.36 0.007037 2.06 8.33E−08 NR_039917 0 chr21 46011639 46011902 + 0.46 0.463277 1.6 8.76E−05 NM_198688 0 chr22 48711353 48712110 + 2.78 4.29E−05 1.81 9.31E−11 NR_036172 0 chr22 48932686 48933292 + 0.89 0.048052 1.78 9.11E−08 NR_038917 0 chr3 13728707 13729763 + 1.18 0.001315 1.84 3.04E−17 NR_027103 0 chr3 13731953 13732599 + 0.87 0.062557 1.68 1.07E−09 NR_027103 0 chr3 59231154 59231995 + 2.04 0.006345 1.99 1.02E−14 NM_198463 0 chr3 64875851 64876079 + 0.19 0.424653 1.75 2.65E−05 NR_030317 0 chr3 86562326 86562565 + 1.63 0.07633 1.62 3.67E−05 NR_046939 0 chr3 86703026 86703274 + 1.63 0.07633 1.67 3.87E−05 NR_046939 0 chr3 1.01E+08 1.01E+08 + 0.59 0.149458 2.36 5.33E−05 NM_014415 0 chr3 1.12E+08 1.12E+08 + −1.03 0.107039 1.68 6.15E−05 NM_183061 0 chr3 1.34E+08 1.34E+08 + 2.26 0.065139 2.68 1.57E−05 NM_178554 0 chr3 1.53E+08 1.53E+08 + −0.54 0.281197 3.46 7.46E−06 NM_001101337 0 chr3 1.97E+08 1.97E+08 + −1.13 0.040692 1.72 4.05E−05 NR_039960 0 chr4 46707646 46707957 + 2.36 0.007037 1.54 1.27E−05 NM_130902 0 chr4 90630842 90631320 + 0.92 0.063595 1.41 7.38E−09 NR_045481 0 chr4 1.26E+08 1.26E+08 + 0.04 0.513686 3.2 1.25E−06 NR_031746 0 chr4 1.56E+08 1.56E+08 + 1.04 0.156144 2.63 2.67E−05 NM_000910 0 chr4 1.56E+08 1.56E+08 + 1.04 0.218994 2.88 1.00E−07 NM_000910 0 chr4 1.58E+08 1.58E+08 + 0.13 0.464479 2.36 5.33E−05 NR_026992 0 chr4 1.89E+08 1.89E+08 + 0.58 0.088758 1.36 4.60E−09 NM_174900 0 chr5  9165629  9165809 + 0.81 0.93262 2.93 2.01E−05 NR_039779 0 chr5  9166378  9167506 + 2.04 1.08E−06 1.56 1.19E−13 NR_039779 0 chr5  9623426  9623573 + −0.54 0.3033 2.68 1.57E−05 NM_019599 0 chr5 16887753 16887906 + −0.22 0.38435 2.58 6.51E−07 NM_012334 0 chr5 86415644 86416216 + 1.87 1.62E−13 2.14 3.90E−10 NR_036243 0 chr5 86421717 86421976 + 1.97 9.83E−07 3.65 2.246−09 NR_036243 0 chr5 86433127 86434310 + 2.84 8.85E−20 1.31 1.95E−10 NR_036243 0 chr5 86484078 86484518 + 2.21 0.00054 1.43 2.13E−07 NR_036249 0 chr5 1.46E+08 1.46E+08 + 3.16 0.004461 1.96 1.02E−08 NM_194251 0 chr5 1.46E+08 1.46E+08 + 0.13 0.429293 1.6 8.76E−05 NR_047115 0 chr5 1.52E+08 1.52E+08 + 2.46 0.042327 3.68 1.25E−09 NM_020167 0 chr5 1.52E+08 1.52E+08 + 2.87 1.79E−05 1.61 1.25E−08 NM_020167 0 chr5 1.54E+08 1.54E+08 + 1.46 0.005935 1.77 4.59E−08 NR_037897 0 chr5  1.6E+08  1.6E+08 + 1.92 0.003755 1.38 6.92E−06 NR_036095 0 chr5  1.7E+08  1.7E+08 + 0.04 0.504309 1.71 2.02E−10 NM_004137 0 chr6 22789703 22789842 + −1.18 0.025593 2.52 7.56E−05 NM_138574 0 chr6 44499354 44499687 + 1.78 0.01992 1.51 1.88E−05 NR_039785 0 chr6 1.32E+08 1.32E+08 + 1.73 0.001876 2.14 2.24E−25 NR_038981 0 chr7 19564732 19564940 + −0.59 0.05591 2.23 2.44E−06 NR_036101 0 chr7 20073788 20073917 + −0.51 0.00454 3.5 2.29E−08 NR_046756 0 chr7 43178657 43179090 + 1.33 0.082052 2.65 3.11E−14 NR_037508 0 chr7 1.28E+08 1.28E+08 + −0.54 0.281197 2.31 8.78E−05 NR_027330 0 chr7 1.38E+08 1.38E+08 + 0.46 0.267559 1.55 2.18E−06 NM_004717 0 chr7 1.43E+08 1.43E+08 + 1.81 4.89E−05 1.78 1.92E−06 NM_001001667 0 chr7 1.56E+08 1.56E+08 + 0.86 0.016783 2.37 2.37E−09 NM_000193 0 chr7 1.57E+08 1.57E+08 + 1.26 0.156387 2.52 7.56E−05 NR_029689 0 chr7 1.57E+08 1.57E+08 + 1.64 1.70E+06 2.56 1.81E−17 NR_029689 0 chr7 1.57E+08 1.57E+08 + 0.26 0.222416 1.83 2.91E−09 NR_029689 0 chr7 1.57E+08 1.57E+08 + −0.13 0.422029 3.1 3.83E−06 NR_029689 0 chr8 23552943 23553115 + −0.54 0.329466 2.12 1.03E−05 NM_001136271 0 chr8 24591737 24592027 + 0.78 0.104428 2.0 5.55E−08 NM_005382 0 chr8 24768615 24769206 + 0.51 0.064611 1.66 1.80E−12 NM_005382 0 chr8 39439535 39439711 + 1.04 0.156144 2.31 8.78E−05 NM_001190956 0 chr8 69547154 69547337 + 2.46 0.004821 3.05 6.54E−09 NR_038877 0 chr8 69552599 69553402 + 1.45 0.000395 2.82 3.25E−20 NR_038877 0 chr8 69610620 69611398 + 3.79 2.08E−27 3.45 8.54E−28 NR_038877 0 chr8 69612680 69612862 + 2.63 0.027294 4.01 1.13E−08 NR_038877 0 chr8 69670588 69870752 + 2.03 1.02E−05 3.2 1.25E−06 NR_039986 0 chr8 69671810 69672516 + 4.28 3.02E−21 2.73 1.31E−18 NR_039986 0 chr8 69698803 69699391 + 2.01 1.78E−05 1.89 5.40E−13 NR_039983 0 chr8 69765707 69766261 + 1.52 0.007411 2.7 6.43E−16 NR_039986 0 chr8 69828895 69829029 + 2.63 0.027294 2.54 6.31E−09 NR_039986 0 chr8 69942535 69943233 + 2.23 2.05E−05 1.72 3.14E−10 NR_039986 0 chr8 69946021 69946257 + 2.63 0.027294 3.68 1.25E−09 NR_039986 0 chr8 70178481 70178636 + 0.81 0.005384 3.63 1.30E−06 NR_039986 0 chr8 70189511 70190321 + 0.78 0.058164 3.22 6.38E−21 NR_039986 0 chr8 1.11E+08 1.11E+08 + 0.97 0.002477 1.38 5.32E−10 NM_014379 0 chr8 1.25E+08 1.25E+08 + 0.45 0.015307 2.55 1.66E−08 NM_001039112 0 chr8 1.29E+08 1.29E+08 + 1.14 0.016598 1.82 1.64E−08 NR_031610 0 chr8 1.43E+08 1.43E+08 + 1.78 1.12E−07 2.55 3.13E−30 NM_207414 0 chr8 1.43E+08 1.43E+08 + 1.12 0.001111 2.4 3.16E−26 NM_207414 0 chr8 1.43E+08 1.43E+08 + 1.87 2.79E−143 1.63 9.63E−180 NM_207414 0 chr8 1.425+08 1.43E+08 + 1.46 0.010305 3.83 1.23E−07 NM_207414 0 chr8 1.43E+08 1.43E+08 + 2.08 1.20E−63 2.55 9.09E−130 NM_207414 0 chr8 1.43E+08 1.43E+08 + 1.7 0.01132 2.88 1.84E−06 NM_207414 0 chr8 1.43E+08 1.43E+08 + 0.87 0.210905 3.58 7.18E−09 NM_207414 0 chr8 1.43E+08 1.43E+08 + 1.33 0.019547 2.96 1.51E−20 NM_207414 0 chr8 1.43E+08 1.43E+08 + 0.86 0.010197 1.53 1.13E−10 NM_207414 0 chr8 1.43E+08 1.43E+08 + 1.48 2.01E−24 1.66 1.10E−49 NM_207414 0 chr8 1.43E+08 1.43E+08 + 0.74 0.000836 1.99 4.61E−28 NM_207414 0 chr8 1.43E+08 1.43E+08 + 1.01 0.005043 1.83 2.21E−10 NM_207414 0 chr8 1.43E+08 1.43E+08 + 0.92 0.013438 2.68 1.20E−10 NM_207414 0 chr8 1.43E+08 1.43E+08 + 1.95 3.98E−11 2.85 1.16E−61 NM_207414 0 chr9 16515973 16518371 + 1.04 0.114476 1.35 3.96E−05 NR_073471 0 chr9 16522104 16522831 + 2.08 9.74E−05 1.67 1.44E−08 NR_073471 0 chr9 73363819 73364036 + 0.94 0.113163 2.03 1.09E−05 NR_029621 0 chr9 78104259 78104394 + 1.78 0.149771 2.31 8.78E−05 NR_031679 0 chr9 92238839 92238986 + 0.45 0.015307 3.29 4.03E−07 NR_024280 0 chr9 92457498 92458145 + 0.98 0.049001 1.98 1.59E−14 NR_924280 0 chr9 92504302 92504458 + 0.87 0.210905 2.5 1.82E−06 NR_024280 0 chr9 92691840 92692890 + 1.3 0.004056 1.62 4.49E−11 NR_036253 0 chr9 92764997 92765272 + 1.33 0.082052 1.52 7.61E−05 NR_036253 0 chr9  1.2E+08  1.2E+08 + −0.36 0.926622 3.58 2.33E−06 NR_003708 0 chr9  1.2E+08  1.2E+08 + 2.04 0.02399 2.72 4.00E−13 NR_003708 0 chr9  1.2E+08  1.2E+08 + 0.46 0.404925 1.99 3.61E−11 NR_003708 0 chr9 1.37E+08 1.37E+08 + 0.4 0.234774 1.72 9.93E−09 NR_039816 0 chr9 1.37E+08 1.37E+08 + 1.2 0.002108 1.3 2.87E−05 NR_039816 0 chrX  1.3E+08  1.3E+08 + 4.29 2.13E−11 2.04 1.68E−09 NM_178471 0 chrX 1.32E+08 1.32E+08 + −2.36 0.648257 3.4 1.33E−05 NR_046891 0 chrX 1.45E+08 1.45E+08 + 1.78 0.052385 2.2 1.53E−05 NR_030581 0 chrY  6003942  6004091 + 1.73 8.24E−05 4.27 6.94E−06 NR_003593 0 chr14 28812828 28812959 + 0.11 0.462988 2.35 9.04E−08 NM_005249 −0.01 chr11 12757237 12757492 + −0.96 0.063073 1.91 7.26E−06 NM_021961 −0.02 chr6  680566  681209 + −0.1 0.404446 1.3 1.97E−07 NM_018303 −0.02 chr6 1.48E+08 1.48E+08 + 0.46 0.404925 2.19 3.14E−07 NM_001030060 −0.02 chr7 55843574 55844292 + 0.96 1.36E−27 1.56 3.47E−46 NR_003949 −0.02 chr1 1.81E+08 1.81E+08 + 0.73 0.017699 1.34 3.23E−06 NM_020950 −0.03 chr12 95804840 95805100 + −0.32 0.326824 2.3 9.22E−07 NM_006838 −0.03 chr19 14669628 14669765 + 0.36 0.303275 3.68 7.23E−07 NM_004146 −0.03 chr22 22177308 22177515 + −1.54 0.060085 2.41 3.22E−05 NM_002745 −0.03 chr3 1.54E+08 1.54E+08 + 1.34 0.000831 1.3 8.05E−05 NM_001114397 −0.03 chr3 1.54E+08 1.54E+08 + −0.22 0.35557 2.58 4.50E−05 NM_001114397 −0.03 chr5 86621733 86621861 + 1.04 0.325105 1.98 2.21E−06 NM_022650 −0.03 chr1 68674563 66674892 + 1.04 0.156144 1.46 6.44E−05 NM_001193334 −0.04 chr12 19609490 19609927 + 1.04 0.06437 1.37 5.70E−07 NM_001267043 −0.04 chr14 78255769 78255979 + 1.46 0.058426 1.78 1.73E−05 NM_020421 −0.04 chr19 39862553 39863647 + 1.77 1.33E−20 1.6 2.23E−16 NM_001256826 −0.04 chr19 56592633 56592902 + −0.32 0.326824 1.78 1.92E−06 NM_001002836 −0.04 chr20 11776581 11776709 + 0.46 0.463277 3.52 4.18E−06 NM_181443 −0.04 chr20 11888084 11888218 + −0.03 0.460616 2.01 4.17E−05 NM_014962 −0.04 chr20 12094211 12094431 + 1.04 0.114478 1.72 4.05E−05 NM_014962 −0.04 chr4 1.52E+08 1.52E+08 + −1.51 1.18E−05 1.71 0.001489 NM_006439 −0.04 chrX 1.28E+08 1.28E+08 + 0.87 0.110781 1.35 1.30E−06 NM_003069 −0.04 chr1 2.01E+08 2.01E+08 + 1.46 0.109973 3.88 6.78E−08 NM_203459 −0.05 chr12 50609599 50609727 + 1.57 0.041179 2.81 5.99E−05 NM_001113547 −0.05 chr15 83480940 83481157 + 2.04 0.02399 1.99 1.72E−05 NM_001080435 −0.05 chr19 18458753 18458995 + 0.46 0.143077 1.9 5.25E−07 NM_017712 −0.05 chr2 1.47E+08 1.47E+08 + 1.16 0.08465 2.58 9.92E−09 NR_026904 −0.05 chr20 25201278 25201452 + −0.13 0.39179 2.23 2.44E−06 NM_001114089 −0.05 chr22 26964283 26964493 + 0.46 0.334119 1.8 6.58E−05 NM_001008566 −0.05 chr7  2847783  2848050 + 0.06 0.121103 1.7 5.53E−08 NM_007353 −0.05 chr1 2.36E+08 2.36E+08 + 0.26 0.362534 4.52 5.98E−07 NM_002508 −0.06 chr10 13987015 13987199 + −1.54 0.103108 2.15 9.64E−05 NM_003675 −0.06 chr12 32362947 32363194 + 0.46 0.208746 1.84 1.76E−05 NM_001714 −0.06 chr12 75940067 75940196 + −3.13 0.009517 3.05 6.68E−06 NM_007043 −0.06 chr14 22723176 22723469 + 1.86 0.000267 1.5 1.86E−06 NM_001344 −0.06 chr14 22838657 22838906 + 1.57 0.041179 1.51 1.88E−05 NM_001344 −0.06 chr19 11012107 11012498 + 1.48 6.30E−05 1.31 2.62E−06 NM_024029 −0.06 chr19 15386558 15386748 + 0.87 0.062557 1.58 2.17E−05 NM_014299 −0.06 chr5 1.51E+08 1.51E+08 + 0.15 0.403369 3.83 1.23E−07 NM_003118 −0.06 chr5 1.71E+08 1.71E+08 + 1.26 0.061907 2.32 1.48E−07 NM_012300 −0.06 chr9 1.25E+08 1.25E+08 + 2.63 0.027294 1.72 4.05E−05 NM_014222 −0.06 chr19 18262164 18262350 + −0.04 0.452279 2.03 1.09E−05 NM_005027 −0.07 chr2 85985751 85986035 + 2.63 0.027294 2.09 4.71E−07 NM_032827 −0.07 chr2 85989124 85989252 + −1.22 0.064934 4.52 5.98E−07 NM_032827 −0.07 chr2 85994046 85994332 + −0.37 0.283497 1.67 7.30E−07 NM_032827 −0.07 chr6 1.06E+08 1.06E+08 + 1.73 8.24E−05 1.35 0.000178 NM_002726 −0.07 chr6 1.06E+08 1.06E+08 + −1.54 0.187264 2.58 4.50E−05 NM_002726 −0.07 chr15 40126126 40126256 + 0.04 0.529972 2.51 1.16E−05 NM_001271855 −0.08 chr5 31413148 31413415 + 0.66 0.121103 2.62 3.68E−08 NM_001100412 −0.08 chrX 1.33E+08 1.33E+08 + 2.93 4.28E−30 2.38 1.90E−19 NR_029523 −0.08 chrX 1.33E+08 1.33E+08 + 2.82 1.27E−20 2.27 4.50E−14 NR_029523 −0.08 chr16 28774465 28774795 + 0.23 0.391248 2.06 7.49E−07 NM_001099651 −0.09 chr1 1.51E+08 1.51E+08 + 1.59 0.009203 1.63 5.84E−05 NM_144618 −0.1 chr16  580679  580827 + −1.28 0.107269 2.78 5.42E−06 NM_005632 −0.1 chr18  4549715  4550035 + −0.13 0.411748 2.57 2.54E−11 NM_004746 −0.1 chr22 32356606 32356897 + 0.87 0.210905 1.76 1.10E−05 NM_015372 −0.1 chr4 1.57E+08 1.57E+08 + 0.18 0.316858 1.81 1.12E−05 NM_016205 −0.1 chr4 1.58E+08 1.58E+08 + 0.36 0.303275 2.11 3.97E−05 NM_016205 −0.1 chr4 1.58E+08 1.58E+08 + −0.16 0.369491 3.23 1.26E−08 NM_016205 −0.1 chr8 1.22E+08 1.22E+08 + 0.75 0.075101 2.11 3.97E−05 NM_002514 −0.1 chr10 45546217 45546405 + 1.04 0.156144 2.29 2.42E−07 NM_001039380 −0.11 chr10 86097700 86097828 + 0.46 0.334119 2.88 3.48E−05 NM_018999 −0.11 chr17 79972458 79972586 + −0.18 0.381896 2.2 5.95E−05 NM_144998 −0.11 chr19 15518848 15518996 + 2.83 2.77E−05 1.66 0.000575 NM_014371 −0.11 chr7 69271595 69271909 + −0.67 0.128489 1.65 9.30E−05 NM_001127232 −0.11 chr18 55836086 55836255 + 1.7 0.01132 2.88 1.84E−06 NM_001144968 −0.12 chr20 45542430 45542609 + 0.56 0.128974 2.05 2.62E−05 NM_172110 −0.12 chr20 45616006 45616165 + −0.54 0.215984 1.65 9.30E−05 NM_172110 −0.12 chr1 88666310 88666438 + 1.46 0.222672 2.25 3.66E−05 NM_006256 −0.13 chr15 29363501 29363677 + 1.81 0.834416 2.81 5.99E−05 NM_005503 −0.13 chr20 17630485 17630744 + −0.39 0.265331 1.52 2.99E−05 NM_001042576 −0.13 chr3 1.97E+08 1.97E+08 + 0.54 0.106535 1.48 4.43E−08 NM_004051 −0.13 chr4 25009191 25009509 + 0.8 0.026275 1.43 5.89E−05 NM_018176 −0.13 chr4 25024621 25024749 + 0.78 0.14308 3.27 4.18E−05 NM_018176 −0.13 chr5 73147145 73147508 + 0.87 0.110781 1.41 211E−05 NM_001244364 −0.13 chr5 73469496 73469771 + −0.3 0.29184 2.98 1.96E−08 NM_001244364 −0.13 chr6 1.12E+08 1.12E+08 + −0.28 0.278449 1.68 4.29E−06 NM_001164281 −0.13 chr1 2.29E+08 2.29E+08 + 0.23 0.391248 1.65 9.30E−05 NM_004578 −0.14 chr17 71433312 71433502 + 1.04 0.156144 3.12 6.79E−08 NM_012121 −0.14 chr17 71461219 71462729 + 2.71 4.82E−22 1.31 4.19E−20 NM_012121 −0.14 chr21 45725078 45725852 + 1.06 0.000573 1.52 9.05E−09 NM_002626 −0.14 chr7 21657845 21657986 + 0.46 0.286689 3.54 1.28E−08 NM_001277115 −0.14 chr9 1.08E+08 1.08E+08 + −0.54 0.22977 1.95 2.71E−05 NM_080546 −0.14 chr9 1.08E+08 1.08E+08 + 1.46 0.222672 2.52 7.56E−05 NM_080546 −0.14 chr11 1.06E+08 1.06E+08 + 0.94 0.113163 2.52 7.56E−05 NM_032424 −0.15 chr2 1.23E+08 1.23E+08 + 0 0.593079 2.19 3.14E−07 NR_048556 −0.15 chr8  5879386  5879562 + −0.13 0.422029 2.01 4.17E−05 NR_040040 −0.15 chr9 1.31E+08 1.31E+08 + 1.04 0.218994 2.78 5.42E−06 NM_001006641 −0.15 chr1 2.35E+08 2.35E+08 + 1.04 0.325105 2.2 5.95E−05 NM_001012985 −0.16 chr11 1.17E+08 1.17E+08 + 0.16 0.427697 1.39 4.86E−05 NM_014956 −0.16 chr8 24019532 24019740 + −0.59 0.05845 1.7 6.77E−06 NM_001142730 −0.16 chr18 24113494 24113630 + 0.56 0.216906 2.42 5.02E−06 NM_001142730 −0.16 chr19 17594685 17594813 + 1.78 0.001315 3.05 6.68E−08 NM_198580 −0.16 chr5 52391142 52391595 + 1.01 0.005043 2.4 3.84E−11 NM_004531 −0.16 chr9 1.34E+08 1.34E+08 + −1.35 0.06335 1.95 2.71E−05 NM_006059 −0.16 chr14 1.06E+08 1.06E+08 + 0.13 0.464479 1.44 1.46E−05 NM_145685 −0.17 chr6 37830297 37830470 + 1.26 0.156387 2.46 1.94E−05 NM_021943 −0.17 chr13 24232830 24232958 + 0.74 0.137564 3.15 2.19E−06 NM_001204458 −0.18 chr2 1.14E+08 1.14E+08 + −0.44 0.196391 1.38 7.74E−05 NM_003466 −0.18 chr2 1.14E+08 1.14E+08 + 0.7 0.016184 1.99 3.68E−08 NM_003466 −0.18 chr2 2.39E+08 2.39E+08 + −0.03 0.460616 1.76 5.85E−08 NM_001137551 −0.18 chr20 55826453 55826700 + 2.54 0.00302 1.4 4.32E−06 NM_001719 −0.18 chr21 46511547 46511770 + −0.54 0.3033 2.03 1.19E−06 NR_027674 −0.19 chr22 50732336 50733389 + 1.94 9.77E−14 1.66 6.00E−15 NM_012401 −0.19 chr22 50734451 50734594 + 4.04 2.38E−05 2.52 7.56E−05 NM_012401 −0.19 chr6 50797659 50797966 + 0.19 0.465306 1.39 3.05E−05 NM_003221 −0.19 chr7 95526342 95526471 + 0.64 0.009079 4.34 3.77E−06 NM_004411 −0.19 chr9 1.37E+08 1.37E+08 + 1.04 0.325105 4.27 6.94E−06 NM_001278074 −0.19 chr9 1.37E+08 1.37E+08 + −0.96 0.104912 1.43 2.13E−07 NM_001278074 −0.19 chr9 1.37E+08 1.37E+08 + 0.23 0.02581 3.2 1.25E−06 NM_001278074 −0.19 chr1 1.55E+08 1.55E+08 + 1.04 0.085353 2.31 8.78E−05 NM_001278230 −0.2 chr18 47019391 47019580 + −0.44 0.196391 1.51 1.88E−05 NM_000985 −0.2 chr19  3548581  3548873 + 0.09 0.430019 1.85 2.21E−07 NM_021731 −0.2 chr3 48750631 48750763 + −1 0.064264 2.15 9.54E−05 NM_001146179 −0.2 chr3 50163676 50163877 + −0.96 0.104912 2.11 3.97E−05 NR_045388 −0.2 chr4 1.19E+08 1.19E+08 + −0.1 0.415961 1.91 1.75E−05 NM_003619 −0.2 chr4 1.19E+08 1.19E+08 + 0.19 0.465306 3.4 1.33E−05 NM_003619 −0.2 chr6 18198227 18198382 + 0.13 0.464479 2.46 1.94E−05 NM_153042 −0.2 chr3  4566203  4566555 + 0.68 0.126482 1.31 5.07E−05 NM_001099952 −0.21 chr3 1.44E+08 1.44E+08 + −0.03 0.460616 1.79 7.19E−06 NM_173653 −0.21 chr3 1.44E+08 1.44E+08 + 0.19 0.424653 1.7 9.76E−05 NM_173653 −0.21 chr4 36028221 36028436 + −0.04 0.956481 3.09 1.18E−07 NM_015230 −0.21 chr8 17577806 17577999 + 1.04 0.085353 1.88 2.74E−05 NM_001001931 −0.21 chr12 29696494 29696805 + 1.78 0.149771 1.7 2.56E−05 NM_183378 −0.22 chr12 75867690 75868349 + 1.16 0.08465 1.96 4.92E−10 NM_006851 −0.22 chr18 13956850 13957098 + 2.3 4.68E−05 1.34 2.25E−05 NM_000529 −0.22 chr11  3066126  3066322 + 0.19 0.396995 2.07 6.37E−05 NM_001014437 −0.23 chr13 28198440 28198931 + 3.46 0.0011 1.41 2.11E−05 NM_152705 −0.23 chr2 1.59E+08 1.59E+08 + 2.92 0.000339 1.84 4.25E−01 NM_001111067 −0.23 chr1 1.66E+08 1.66E+08 + −2.36 0.648257 2.83 3.17E−06 NM_001017961 −0.24 chr1 1.66E+08 1.66E+08 + 0.65 0.173721 1.48 1.74E−07 NM_001017961 −0.24 chr10 1.26E+08 1.26E+08 + 0.22 0.351988 2.64 2.45E−06 NM_014661 −0.24 chr17 17744110 17744557 + 0.05 0.422088 1.56 4.49E−00 NM_001005291 −0.24 chr19  6063761  6063815 + −0.28 0.353275 1.91 1.75E−05 NM_134433 −0.24 chr7 1.32E+08 1.32E+08 + −0.17 0.314291 1.42 3.16E−12 NM_001105543 −0.24 chr7 1.32E+08 1.32E+08 + 0.73 0.00011 1.42 1.57E−28 NM_001105543 −0.24 chr7 1.32E+08 1.32E+08 + 0.44 0.077846 1.53 7.95E−20 NM_001105543 −0.24 chr7 1.32E+08 1.32E+08 + 2.26 0.065139 3.05 6.68E−06 NM_001105\543 −0.24 chrX 1.53E+08 1.53E+08 + 1.46 0.018075 1.7 9.76E−05 NM_001105543 −0.24 chr17 19549682 19549816 + −0.3 0.29184 2.41 3.22E−05 NM_000382 −0.25 chr5 1.37E+08 1.37E+08 + 0.78 0.301098 2.31 8.78E−05 NM_004598 −0.25 chr5 1.41E+08 1.41E+08 + 0.89 0.028357 1.79 3.37E−07 NM_005471 −0.25 chr9  734802  735094 + 1.01 0.003957 1.46 4.04E−05 NM_153186 −0.25 chr10 88161336 88161490 + 1.46 0.222672 1.72 4.05E−05 NM_017551 −0.26 chr17 19212355 19213080 + 0.54 0.009121 1.46 1.08E−09 NR_048576 −0.26 chr3 64627450 64627632 + 2.11 1.94E−05 2.07 6.37E−05 NR_038264 −0.26 chr3 64628321 64628647 + 1.96 0.000304 1.58 2.17E−05 NR_038264 −0.26 chr3 64644888 64645413 + 1.29 0.006935 1.88 1.43E−07 NR_038264 −0.26 chr4 54872591 54873820 + 2.33 6.23E−28 1.66 4.42E−13 NR_027153 −0.26 chr4 54877404 54877676 + 3.02 2.18E−20 1.85 3.03E−06 NR_027153 −0.26 chr4 54878290 54878448 + 2.31 5.03E−08 2.46 3.03E−06 NR_027153 −0.26 chr12  1.2E+08  12E+08 + 0.78 0.301098 2.63 2.67E−05 NM_001136534 −0.27 chr19 45608097 45608261 + 0.57 0.230388 2.06 6.83E−06 NM_019121 −0.27 chr14 1.05E+08 1.05E+08 + 0.81 0.079776 2.25 3.66E−05 NM_015656 −0.28 chr14 1.05E+08 1.05E+08 + 0.46 0.308555 3.4 1.33E−05 NM_015656 −0.28 chr2 84609937 84610232 + 0.46 0.167227 2.12 2.95E−07 NM_003849 −0.28 chr8 1.24E+08 1.24E+08 + 0.51 0.147807 1.37 1.14E−10 NM_014943 −0.28 chr14 49440307 49440548 + −0.74 0.042426 1.44 1.46E−05 NM_001030001 −0.29 chr15 38807024 38807199 + 0.04 0.529972 2.6 4.14E−06 NM_001128602 −0.29 chr7 23500778 23500940 + −0.2 0.353044 1.75 2.65E−05 NM_006547 −0.29 chr8 1.07E+08 1.07E+08 + 0.59 0.149458 2.75 4.74E−08 NM_018002 −0.29 chr11 1.13E+08 1.13E+08 + 0.92 0.152933 1.8 6.58E−05 NR_034101 −0.3 chr14 72192384 72192771 + 0.37 0.15055 1.34 1.42E−05 NM_015556 −0.3 chr13 61330417 61330978 + 0.36 0.303275 1.44 3.67E−07 NM_001146070 −0.32 chr19 13215124 13215269 + −0.16 0.376068 2.35 2.16E−06 NM_005583 −0.32 chr2 68642008 68642167 + −0.54 0.361994 3.05 6.68E−06 NM_002664 −0.32 chr4 54153510 54153789 + 1.6 0.002197 2.95 3.39E−08 NM_152540 −0.32 chr4  1.1E+08  1.1E+08 + −2.13 0.027712 2.58 4.50E−05 NM_001256074 −0.32 chr4 1.43E+08 1.43E+08 + 2.16 0.016043 1.65 2.44E−05 NM_003866 −0.32 chr4 1.43E+08 1.43E+08 + −0.2 0.32972 1.68 1.62E−05 NM_003866 −0.32 chr4 1.44E+08 1.44E+08 + −0.13 0.422029 1.37 2.50E−06 NM_003866 −0.32 chr5 1.39E+08 1.39E+08 + −1.36 0.791388 1.95 1.12E−05 NM_032289 −0.32 chr10 52336134 52336787 + 0.78 0.14308 1.3 3.09E−07 NM_147156 −0.33 chr16  4441454  4441968 + 1.2 0.002108 2.11 2.65E−10 NM_138440 −0.34 chr2 74112768 74113003 + 0.29 0.337406 1.59 5.50E−05 NM_001199893 −0.35 chr5 52159831 52160154 + 0.33 0.277101 2.32 3.28E−12 NM_181501 −0.35 chr5 52164262 52164394 + 0.04 0.513686 3.12 6.79E−08 NM_181501 −0.35 chr1 1.75E+08 1.75E+08 + 1.46 0.109973 2.34 5.64E−07 NM_003285 −0.36 chr22 40795782 40795944 + 0.85 0.021439 2.05 2.62E−05 NM_015705 −0.36 chr3 62234917 62235103 + −0.13 0.422029 2.68 1.57E−05 NR_038283 −0.38 chr10 60726012 60726214 + −0.06 0.44608 2.73 9.25E−06 NM_032439 −0.41 chr10 60885594 60885936 + 0.45 0.015307 1.78 8.03E−07 NM_032439 −0.41 chr13 41172002 41172134 + −2.36 0.648257 3.73 4.01E−07 NM_002015 −0.42 chr3 13142294 13142539 + 0.33 0.277101 1.59 5.50E−05 NM_001134382 −0.42 chr1 15359923 15360135 + −0.96 0.18457 1.46 6.44E−05 NM_001017999 −0.43 chr2 1.12E+08 1.12E+08 + 0.13 0.429293 1.68 6.15E−05 NR_024373 −0.44 chr8 17626529 17626682 + 2.04 0.099329 2.6 4.14E−06 NM_001001924 −0.45 chr4 93021108 93021236 + 2.04 0.099329 3.34 2.36E−05 NM_001510 −0.46 chr4 93255689 93255904 + 0.45 0.015307 3.5 2.29E−08 NM_001510 −0.46 chr13 39210948 39211076 + −2.13 0.027712 3.52 4.18E−06 NM_207361 −0.46 chr5 1.46E+08 1.46E+08 + 1.2 0.002108 1.86 2.32E−13 NM_181675 −0.49 chr5 1.46E+08 1.46E+08 + 1.78 0.149771 2.61 3.88E−07 NM_181675 −0.49 chr5 1.46E+08 1.46E+08 + 2.54 0.00302 2.22 1.17E−08 NM_181675 −0.49 chr5 1.46E+08 1.46E+08 + 1.55 0.000234 1.9 1.63E−08 NM_181675 −0.49 chr5 1.46E+08 1.46E+08 + 0.78 0.201565 1.51 1.88E−05 NM_181675 −0.49 chr5 1.46E+08 1.46E+08 + 2.63 0.027294 3.15 2.19E−06 NM_181675 −0.49 chr8 64478362 64478992 + 2.49 7.99E−07 1.75 1.18E−09 NR_038875 −0.49 chr2  1.9E+08  1.9E+08 + 0.78 0.301098 2.15 9.64E−05 NM_014585 −0.5 chr11 1.32E+09 1.32E+08 + 1.29 5.83E−05 1.72 8.19E−18 NM_018522 −0.51 chr11 1.32E+08 1.32E+08 + −0.24 0.279739 2.58 4.50E−05 NM_016522 −0.51 chr11 44584566 44584885 + −0.98 0.015887 1.72 1.19E−06 NM_002231 −0.52 chr14 1.03E+08 1.03E+08 + 0.11 0.462988 2.25 3.66E−05 NR_073542 −0.53 chr1 31221034 31222083 + 1.57 1.65E−06 1.63 9.96E−19 NM_006762 −0.55 chr1 31277302 31277909 + −0.78 0.010359 1.37 2.17E−08 NM_006762 −0.55 chr10 62052666 62053455 + −0.98 0.010199 1.48 4.43E−08 NM_020987 −0.55 chr10 62288823 62288970 + 0.98 0.049001 2.41 3.22E−05 NM_001204404 −0.55 chr8 93065927 93066564 + 1.7 0.01132 2.04 1.68E−09 NM_004349 −0.55 chr10 61099144 61099392 + 1.19 0.036046 2.61 5.72E−10 NM_001166698 −0.56 chr8 93146827 93146961 + 0.04 0.529972 2.78 5.42E−06 NM_01198633 −0.56 chr18 74792841 74792969 + −0.13 0.422029 2.73 9.25E−06 NM_001025100 −0.58 chr3 30375463 30375667 + −0.54 0.3033 2.35 2.16E−06 NM_001024847 −0.58 chr3 30683740 30683988 + 1.26 0.961907 1.99 1.72E−05 NM_001024847 −0.58 chr3 30703324 30703592 + −0.06 0.442247 1.88 1.13E−05 NM_001024847 −0.58 chr4 1.41E+08 1.41E+08 + −1.13 0.188905 2.52 7.56E−05 NM_001204367 −0.58 chr5 62424424 62424557 + −0.36 0.926622 3.63 1.30E−06 NM_181506 −0.58 chr5 62507265 62507441 + 2.26 0.065139 3.42 7.25E−08 NM_181506 −0.58 chr6 1.32E+08 1.32E+08 + 0.46 0.404925 2.34 1.37E−05 NM_001901 −0.58 chrX 1.45E+08 1.45E+08 + 1.83 0.000917 2.15 1.70E−14 NM_032539 −0.58 chrX 1.45E+08 1.45E+08 + 2.26 0.010657 3.46 4.08E−08 NM_032539 −0.58 chrX 1.45E+08 1.45E+08 + 1.26 0.156387 3.42 7.25E−08 NM_001144009 −0.58 chr1 1.87E+08 1.87E+08 + 0.46 0.404925 2.2 1.53E−05 NM_024420 −0.59 chr1 1.87E+08 1.87E+08 + −1.96 0.01731 2.35 9.04E−08 NM_024420 −0.59 chr2 1.83E+08 1.83E+08 + 2.04 0.009329 2.2 1.53E−05 NM_001258314 −0.63 chr22 41057211 41058340 + 1.5 9.85E−06 1.84 1.99E−17 NM_005297 −0.65 chr22 41072122 41072268 + 1.54 0.022876 3.1 3.83E−06 NM_005297 −0.65 chr10 98909413 98909545 + −1.77 0.032657 2.58 4.50E−05 NM_003061 −0.68 chr16 66300679 66300853 + −0.54 0.261994 2.36 5.33E−05 NM_001795 −0.69 chr13 46697061 46697369 + 2.26 0.010657 1.72 1.19E−06 NM_001278541 −0.74 chr20 57936385 57936687 + 0.52 0.173082 1.59 9.83E−07 NM_207034 −0.74 chr17 77021745 77021873 + −0.99 0.000752 2.2 5.95E−05 NR_049769 −0.75 chr2 14759214 14759421 + −0.22 0.38435 2.07 6.37E−06 NM_145175 −0.75 chr2 15050407 15050805 + 0.84 0.035899 1.54 2.41E−08 NM_145175 −0.75 chr14 32964091 32964219 + 0.72 0.192023 2.39 1.31E−06 NM_004274 −0.84 chr6 1.29E+08 1.29E+08 + 2.95 2.62E−07 3.12 2.51E−14 NM_001135648 −0.86 chr1 42385966 42386126 + 0.54 0.204578 2.31 8.78E−05 NR_038260 −0.94 chr16 81440812 81440979 + −1.13 0.188905 2.58 4.50E−05 NR_039871 −0.94 chr18  3989314  3989651 + 0.16 0.264283 1.46 1.60E−07 NR_102

−0.97 chr11 92593088 92593216 + 0.23 0.391248 2.31 5.99E−05 NM_005959 −1 chr13 1.02E+08 1.02E+08 + 0.61 0.157103 1.5 4.71E−07 NR_047687 −1 chr14 87618888 87619016 + 0.19 0.465306 2.31 8.78E−05 NR_038445 −1 chr15 92887156 92887317 + 1.81 0.834416 2.31 8.78E−05 NM_006011 −1 chr4 1.89E+08 1.89E+08 + 1.76 1.97E−06 1.83 3.41E−07 NR_033869 −1 chr4 1.89E+08 1.89E+08 + −1.36 0.791388 2.63 2.67E−05 NR_033869 −1 chr4 1.89E+08 1.89E+08 + 0.19 0.424653 1.54 5.74E−05 NR_033869 −1 chr4 1.89E+08 1.89E+08 + 0.84 0.004799 2.07 7.65E−11 NR_033869 −1 chr5 71837480 71837925 + 0.07 0.454457 1.48 6.87E−07 NM_152625 −1 chr6 35013917 35014257 + −0.35 0.30382 1.9 1.63E−09 NM_001261821 −1 chr8 1.02E+08 1.02E+08 + −0.54 0.215984 1.51 1.88E−05 NM_152628 −1 chr5  1.4E+08  1.4E+09 + 0.78 0.14308 1.61 4.19E−07 NM_031883 −1.05 chr20 49712632 49712903 + 3.04 0.007065 1.37 2.76E−05 NM_002237 −1.11 chr20 49737398 49738692 + 0.12 0.372164 1.67 1.18E−18 NM_002237 −1.11 chr6 1.07E+08 1.07E+08 + 0.78 0.301098 3.4 1.23E−05 NM_182907 −1.12 chr1 1.61E+08 1.61E+08 + 0.04 0.529972 3.2 7.38E−05 NM_021642 −1.13 chr2 30253886 30254215 + 0.46 0.404925 1.37 1.74E−05 NM_004304 −1.14 chr18 31537374 31537520 + 1.36 0.044821 2.1 4.28E−06 NM_001198549 −1.2 chr6 1.15E+08 1.15E+08 + 1.63 0.07633 2.38 8.30E−06 NM_153612 −1.21 chr11 17834821 17835242 + 1.19 0.113922 1.68 1.25E−07 NM_001112741 −1.22 chr11 17895454 17895831 + 0.19 0.396995 1.38 7.74E−05 NM_001112741 −1.22 chr8 24924756 24924894 + 1.46 0.003445 2.51 1.16E−05 NM_006158 −1.22 chr3  1.6E+08 1.6E4+08 + −0.32 0.326824 1.53 5.05E−06 NM_001168214 −1.29 chr3  1.6E+08  1.6E+08 + −1.28 0.107269 1.81 2.71E−05 NM_001168214 −1.29 chr3  1.6E+08  1.6E+08 + 2.83 4.18E−13 1.56 4.49E−09 NM_001168214 −1.29 chr5  1.5E+08  1.5E+08 + 1.55 0.000265 1.37 1.19E−09 NM_001166208 −1.3 chrX 37463206 37463541 + 1.28 0.061907 1.7 2.05E−07 NM_001170331 −1.32 chr7 1.51E+08 1.51E+08 + 0.34 0.120777 1.32 1.94E−16 NM_024429 −1.33 chr7 1.51E+08 1.51E+08 + −0.54 0.329466 2.15 9.64E−05 NM_024429 −1.33 chr11  8389877  8390012 + 0.94 0.113163 2.81 5.99E−05 NM_001270428 −1.4 chr17 10018213 10018475 + 0.26 0.418658 2.03 1.09E−05 NM_201432 −1.44 chr19  6801588  6802052 + −1.35 0.004025 1.66 1.25E−07 NM_005428 −1.46 chr4 96134788 96135552 + 0.72 0.053006 1.75 1.18E−09 NM_001256794 −1.46 chrX  9874995  9875137 + −0.04 0.452279 2.31 8.78E−05 NM_001195081 −1.49 chr17 17651960 17652696 + 1.6 6.92E−11 1.59 2.90E−12 NR_024007 −1.58 chr17 17664127 17664362 + 1.83 0.000917 2.07 1.98E−07 NR_024007 −1.58 chr17 17669266 17669463 + 1.82 0.000383 1.72 4.46E−06 NR_024007 −1.58 chrX 1.45E+08 1.45E+08 + 1.26 0.026644 2.83 1.67E−14 NM_004709 −1.58 chrX 1.45E+08 1.45E+08 + 1.98 0.002571 1.65 2.44E−05 NM_004709 −1.58 chrX 1.45E+08 1.45E+08 + 0.46 0.365076 4.2 1.27E−05 NM_004709 −1.58 chrX 1.45E+08 1.45E+08 + 2.26 0.010657 2.68 1.24E−09 NM_004709 −1.58 chrX 1.45E+08 1.45E+08 + 1.46 0.010305 2.15 9.64E−05 NM_004709 −1.58 chr11 12101852 12102125 + −0.39 0.265331 2.34 5.64E−07 NM_014632 −1.59 chr11 12111840 12112001 + −0.54 0.261994 2.25 3.66E−05 NM_014632 −1.59 chr3  1.9E+08  1.9E+08 + −0.13 0.411748 4.92 4.20E−09 NM_018192 −1.6 chr5 57312368 57312870 + 2.81 8.58E−08 1.89 5.16E−10 NM_001252226 −1.61 chr5 57313486 57314877 + 1.28 5.32E−06 1.48 3.60E−12 NM_001252226 −1.61 chr5 57315615 57316268 + 2.85 4.58E−12 1.4 2.62E−08 NM_001252226 −1.61 chr5 57418112 57418362 + 0.04 0.529972 1.8 6.58E−05 NM_001252226 −1.61 chr5 57724924 57725156 + 1.46 0.109973 3.27 7.19E−09 NM_001252226 −1.61 chr7  4860643  4860771 + −0.54 0.281197 1.8 6.58E−05 NR_039800 −1.69 chr17 53802711 53802839 + 1.26 0.061907 2.2 5.95E−05 NM_018286 −1.75 chr6 1.19E+08 1.19E+08 + −0.77 0.876277 2.31 8.78E−05 NR_037662 −1.81 chr10 45631266 45631624 + 2.29 7.49E−08 1.36 1.43E−06 NR_024472 −2 chr21 42274009 42274387 + −0.41 0.248943 2.25 7.23E−09 NR_073202 −2.02 chr7 32317511 32317647 + −0.54 0.281197 2.34 1.37E−05 NM_001191058 −2.05 chr1 1.63E+08 1.63E+08 + 0.04 0.529972 2.2 1.88E−08 NM_001113380 −2.09 chr3 11042532 11042833 + −0.13 0.411748 1.53 3.19E−06 NM_003042 −2.09 chr7 32097768 32097923 + 1.64 0.000139 3.08 3.76E−09 NM_001191056 −2.28 chr1 17447421 17447579 + 0.46 0.463277 1.92 4.25E−05 NM_007365 −2.32 chr10 1.35E+08 1.35E+08 + −0.28 0.310976 2.93 2.01E−05 NM_015722 −2.32 chr20 57121050 57121600 + −0.2 0.32972 2.02 8.33E−14 NR_034147 −2.43 chr16 82667638 82666111 + 0.8 0.001044 1.98 1.55E−08 NM_001220491 −2.5 chr1 89788157 89788474 + 1.26 0.061907 1.39 3.05E−05 NR_077244 −3.32 chr1 90870532 90870713 + −1.54 0.008491 2.1 4.28E−06 NM_020063 −3.32 chr1 1.81E+08 1.81E+08 + −1.2 0.001262 1.54 1.05E−20 NM_000721 −3.32 chr11 59961362 59961523 + 0.56 0.216906 2.16 2.47E−05 NM_001247999 −3.32 chr11 1.11E+08 1.11E+08 + 0.3 0.381963 1.95 1.12E−05 NM_198498 −3.32 chr11 1.11E+08 1.11E+08 + 0.46 0.404925 2.2 5.95E−05 NM_198498 −3.32 chr11 1.11E+08 1.11E+08 + −2.36 0.648257 3.29 4.03E−07 NM_198498 −3.32 chr11 1.11E+08 1.11E+08 + 2.4 4.44E−07 1.92 0.000104 NM_198498 −3.32 chr16 55314913 55315050 + −1.19 0.000306 2.52 7.56E−05 NM_024335 −3.32 chr18 18847557 18848081 + 1.53 0.01294 2.91 6.64E−17 NM_001142966 −3.32 chr18 18943441 18943744 + 0.46 0.334119 2.01 2.08E−07 NM_001142966 −3.32 chr18 52755385 52755665 + −0.2 0.353044 1.9 2.20E−07 NM_025214 −3.32 chr18 57883581 57885484 + 1.86 6.07E−06 1.86 2.42E.34 NM_005912 −3.32 chr19 43624425 43626178 + 2.45 2.82E−42 1.31 1.28E−20 NM_031246 −3.32 chr2 1.16E+08 1.16E+08 + 0.63 0.261697 1.6 8.76E−05 NM_001004360 −3.32 chr20 42567918 42588208 + −1.54 0.103108 2.54 2.67E−09 NM_001098798 −3.32 chr18 45846339 45846473 + 1.78 0.149771 2.2 5.95E−05 NM_001039360 −3.49 chr13 43467628 43468040 + 0 0.593079 1.9 5.25E−07 NM_001002264 −4.32 chr2 76894792 76894947 + 0.04 0.529972 2.31 8.78E−05 NM_001134745 −4.32 chr5 1.74E+08 1.74E+08 + 2.04 0.000496 1.83 1.23E−09 NM_002449 −4.32 chr5 1.74E+08 1.74E+08 + 0.04 0.529972 2.24 9.42E−06 NM_002449 −4.32 chr5 1.74E+08 1.74E+08 + −1.28 0.107269 2.2 5.95E−05 NM_002449 −4.32 chr19 18875178 18875362 + 0.13 0.464479 1.49 4.40E−05 NM_000095 −4.91 chr22 28602385 28602897 + 0.87 0.062557 1.37 6.27E−06 NR_036169 −6.32

indicates data missing or illegible when filed

Example 9 Effects of p300/CBP HAT Inhibitors

To investigate the effects on MGMT expression in glioblastoma (GBM) cells by the inhibition of the enhancer, GBM cells (3080) were treated with two different p300/CBP HAT inhibitors, CBP1 and CPI-329. Both of them resulted in reduced levels of H3K27ac and reduced expression of MGMT (FIGS. 14A and 14B). The reduced expression levels of MGMT after each treatment were further confirmed by RT-qPCR (FIGS. 15A and 15B).

To test the sensitivity of GBM cells to TMZ, both neurosphere formation assay and colony formation assay were employed. GBM cells (3080) treated with p300/CBP HAT inhibitors (CBP1 and CPI-329) resulted in increased sensitivity to TMZ (FIGS. 16A and 16B).

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All documents cited in this application are hereby incorporated by reference as if recited in full herein.

Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention. 

What is claimed is:
 1. A method for treating or ameliorating the effects of a cancer in a subject, comprising administering to the subject a therapeutically effective amount of a first agent that modulates the activity of an enhancer and a therapeutically effective amount of a second agent that is used to treat the cancer.
 2. The method of claim 1, wherein the enhancer regulates the expression of O₆-methylguanine-DNA-methyltransferase (MGMT) and/or Ki67 in the subject.
 3. The method of claim 1, wherein the first agent is selected from a Bromodomain inhibitor (BETi), a histone acetyltransferase inhibitor (HATi), and combinations thereof.
 4. The method of claim 3, wherein the Bromodomain inhibitor (BETi) is selected from the group consisting of JQ1, I-BET151/762, PF-1, RVX-208, BMS-986158, OTX015, PLX-51107, GSK525762, INCB054329, TEN-010, GSK2820151, BAY 1238097, and combinations thereof.
 5. The method of claim 3, wherein the histone acetyltransferase inhibitor (HATi) is selected from the group consisting of C646, NU-9056, PU141, EML425, L002, MB-3, CPTH2, Plumbagin, Embelin, EGCG, Curcumin, HAT inhibitor II, Garcinol, Anacardic acid, MG 149, Gossypol, CTK7A, Windorphen, LoCAM, TH1834, CTx-1, Lys-CoA, and combinations thereof.
 6. The method of claim 3, wherein the histone acetyltransferase inhibitor (HATi) is a p300 HAT inhibitor.
 7. The method of claim 1, wherein the second agent is temozolomide (TMZ).
 8. The method of claim 7, wherein the subject is TMZ-resistant.
 9. The method of claim 1, wherein the subject is a mammal.
 10. The method of claim 9, wherein the mammal is a human.
 11. The method of claim 1, wherein the cancer is selected from breast cancer, lung cancer, and brain tumor.
 12. The method of claim 11, wherein the brain tumor is gliobastoma (GBM).
 13. The method of claim 1, further comprising administering to the subject a therapeutically effective amount of a histone deaceylase inhibitor (HDACi) that is selected from the group consisting of Trichostatin A, SAHA, Belinostat, Panabiostat, Givinostat, Resminostat, Abexinostat, Quisinostat, Rocilinostat, Practinostat, CHR-3996, Valproic acid, Butyric acid, Phenylbutyric acid, Entinostat, Tacedinaline, 4SC202, Mocetinostat, Rom idepsin, Nicotinamide, Sirtinol, Cambinol, EX-527, and combinations thereof.
 14. A method for delaying the emergence of temozolomide (TMZ) resistance and/or increasing the TMZ sensitivity in a subject, comprising administering to the subject a therapeutically effective amount of an agent that modulates the activity of an enhancer.
 15. The method of claim 14, wherein the enhancer regulates the expression of O₆-methylguanine-DNA-methyltransferase (MGMT) in the subject.
 16. The method of claim 14, wherein the agent is selected from a Bromodomain inhibitor (BETi), a histone acetyltransferase inhibitor (HATi), and combinations thereof.
 17. The method of claim 16, wherein the Bromodomain inhibitor (BETi) is selected from the group consisting of JQ1, I-BET151/762, PF-1, RVX-208, BMS-986158, OTX015, PLX-51107, GSK525762, INCB054329, TEN-010, GSK2820151, BAY 1238097, and combinations thereof.
 18. The method of claim 16, wherein the histone acetyltransferase inhibitor (HATi) is selected from the group consisting of C646, NU-9056, PU141, EML425, L002, MB-3, CPTH2, Plumbagin, Embelin, EGCG, Curcumin, HAT inhibitor II, Garcinol, Anacardic acid, MG 149, Gossypol, CTK7A, Windorphen, LoCAM, TH1834, CTx-1, Lys-CoA, and combinations thereof.
 19. The method of claim 16, wherein the histone acetyltransferase inhibitor (HATi) is a p300 HAT inhibitor.
 20. A method for reducing tumor aggressiveness in a subject, comprising administering to the subject a therapeutically effective amount of an agent that modulates the activity of an enhancer.
 21. The method of claim 20, wherein the enhancer regulates the expression of Ki67 in the subject.
 22. The method of claim 20, wherein the agent is selected from a Bromodomain inhibitor (BETi), a histone acetyltransferase inhibitor (HATi), and combinations thereof.
 23. The method of claim 22, wherein the Bromodomain inhibitor (BETi) is selected from the group consisting of JQ1, I-BET151/762, PF-1, RVX-208, BMS-986158, OTX015, PLX-51107, GSK525762, INCB054329, TEN-010, GSK2820151, BAY 1238097, and combinations thereof.
 24. The method of claim 22, wherein the histone acetyltransferase inhibitor (HATi) is selected from the group consisting of C646, NU-9056, PU141, EML425, L002, MB-3, CPTH2, Plumbagin, Embelin, EGCG, Curcumin, HAT inhibitor II, Garcinol, Anacardic acid, MG 149, Gossypol, CTK7A, Windorphen, LoCAM, TH1834, CTx-1, Lys-CoA, and combinations thereof.
 25. The method of claim 22, wherein the histone acetyltransferase inhibitor (HATi) is a p300 HAT inhibitor.
 26. A pharmaceutical composition comprising 1) a therapeutically effective amount of a first agent that modulates the activity of an enhancer in a subject suffering from a cancer and 2) a therapeutically effective amount of a second agent that is used to treat the cancer.
 27. The pharmaceutical composition of claim 26, wherein the enhancer regulates the expression of O₆-methylguanine-DNA-methyltransferase (MGMT) and/or Ki67 in the subject.
 28. The pharmaceutical composition of claim 26, wherein the first agent is selected from a Bromodomain inhibitor (BETi), a histone acetyltransferase inhibitor (HATi), and combinations thereof.
 29. The pharmaceutical composition of claim 28, wherein the Bromodomain inhibitor (BETi) is selected from the group consisting of JQ1, I-BET151/762, PF-1, RVX-208, BMS-986158, OTX015, PLX-51107, GSK525762, INCB054329, TEN-010, GSK2820151, BAY 1238097, and combinations thereof.
 30. The pharmaceutical composition of claim 28, wherein the histone acetyltransferase inhibitor (HATi) is selected from the group consisting of C646, NU-9056, PU141, EML425, L002, MB-3, CPTH2, Plumbagin, Embelin, EGCG, Curcumin, HAT inhibitor II, Garcinol, Anacardic acid, MG 149, Gossypol, CTK7A, Windorphen, LoCAM, TH1834, CTx-1, Lys-CoA, and combinations thereof.
 31. The pharmaceutical composition of claim 28, wherein the histone acetyltransferase inhibitor (HATi) is a p300 HAT inhibitor.
 32. The pharmaceutical composition of claim 26, wherein the second agent is temozolomide (TMZ).
 33. The pharmaceutical composition of claim 32, wherein the subject is TMZ-resistant.
 34. The pharmaceutical composition of claim 26, wherein the subject is a mammal.
 35. The pharmaceutical composition of claim 34, wherein the mammal is a human.
 36. The pharmaceutical composition of claim 26, wherein the cancer is selected from breast cancer, lung cancer, and brain tumor.
 37. The pharmaceutical composition of claim 36, wherein the brain tumor is gliobastoma (GBM).
 38. The pharmaceutical composition of claim 26, further comprising 3) a therapeutically effective amount of a histone deaceylase inhibitor (HDACi) that is selected from the group consisting of Trichostatin A, SAHA, Belinostat, Panabiostat, Givinostat, Resminostat, Abexinostat, Quisinostat, Rocilinostat, Practinostat, CHR-3996, Valproic acid, Butyric acid, Phenylbutyric acid, Entinostat, Tacedinaline, 4SC202, Mocetinostat, Rom idepsin, Nicotinamide, Sirtinol, Cambinol, EX-527, and combinations thereof.
 39. A kit comprising the pharmaceutical composition according to any one of claims 26-38, and instructions of use.
 40. A method for treating or ameliorating the effects of gliobastoma (GBM) in a subject that is resistant to temozolomide (TMZ), comprising administering to the subject a therapeutically effective amount of a p300 HAT inhibitor or a Bromodomain inhibitor (BETi) and a therapeutically effective amount of TMZ.
 41. A method of modulating proliferation of brain tumor cells with or without activation of a MGMT enhancer, the method comprising contacting the cells with an effective amount of a composition selected from a HAT inhibitor, a p300 inhibitor, a bromodomain inhibitor, and combinations thereof. 